Inhibition of selectin binding

ABSTRACT

This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10 6  fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part during work partially supported by theU.S. Department of Energy under contract DE-AC03-76SF00098. Thegovernment has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of pending U.S. patent application Ser.No. 08/807,428, filed Feb. 28, 1997, which claims priority benefit ofU.S. provisional application No. 60/012,894, filed Mar. 1, 1996, both ofwhich are hereby incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates generally to the field of therapeutic compoundsdesigned to interfere between the binding of carbohydrate ligands andtheir receptors on cell surface. More specifically, it providescompositions of materials whose purpose is inhibiting cell migration andactivation via P- and L-selectin, using polymerized glycoliposomes.

BACKGROUND

The adhesion of circulating neutrophils to endothelial cells is one ofthe important events occurring in the process of inflammation.Neutrophil recruitment to tissues is initiated by an adhesion cascade.Through this process, cells roll and eventually attach firmly to theendothelium. The factors that contribute to the high binding strength ofthis interaction are not fully understood, but is thought to involveinteraction between selectins on one cell with carbohydrate ligands onanother cell. By interfering with the binding between these components,it may be possible to counter pathological sequelae related to cellmigration.

A number of adhesion molecules mediate the interaction of neutrophilsand other leukocytes to the endothelium. Amongst them are the ICAMs,VCAM, CD11, CD18, the integrin I4ν1, and several receptors now knowncollectively as selecting. Each of these molecules is part of aligand-receptor pair, one of which is expressed on each of the twointeracting cells. For a general review, the reader is referred toBevilacqua (Annu. Rev. Immunol. 11:767, 1993). In various combinations,these and other molecules support leukocyte adhesion to the vessel walland extravasation, and may also participate in activation of celleffector functions. Expression of many of these molecules isup-regulated by soluble factors such as cytokines, thereby acting toincrease the recruitment of leukocytes to an affected area.

Amongst the plurality of adhesion molecules that have been described,three have been collected together in a category known as selecting. Onewas formerly known as ELAM-1, and was identified using inhibitorymonoclonal antibodies against cytokine-activated endothelial cells.Another was formerly designated as PADGEM, GMP-140, or CD61. It wasoriginally identified on platelets, and is now known as P-selectin. Athird identified on lymphocytes was formerly designated as mLHR, Leu8,TQ-1, gp90^(MEL), Lam-1, or Lecam-1, and is now known as L-selectin. Theselectins were grouped together on the basis of a structural similarity,before very much was known about their binding specificity. All aresingle chain polypeptides having a carbohydrate binding domain near theN-terminus, an EGF repeat, and anywhere between 2 to 9 modules of ˜60amino acids each sharing homology with complement binding proteins. Forgeneral reviews, the reader is referred to Lasky (Annu. Rev. Biochem.64:113, 1995) and Kansas (Blood 88:3259, 1996).

The three selectins differ from each other in a number of importantrespects. As depicted in FIG. 2A, the selectins have different ligandcounterparts in the adhesion process. Each selectin is regulateddifferently, and participates in a different manner in the process ofinflammation or immunity. There is also an increasing appreciation fordifferences in the ligand binding requirements between the selecting.

E-selectin has garnered a significant amount of recent research interestbecause of its role in inflammation. The migration of inflammatorymediator cells to an inflammatory site is thought to be mediated in partby adhesion of the cells to vascular endothelial cells. Studies in vitrohave suggested that E-selectin participates in the adhesion of not onlyneutrophils, but also eosinophils, monocytes and a subpopulation ofmemory T-cells to endothelium that has been activated by endotoxin,IL-1, or TNF. Expression of E-selectin by endothelial monolayerincreases by about 10-fold and peaks at about 4 hours after stimulationwith IL-1, subsiding to near basal levels within 24 hours. Thebiological role of E-selectin is thought to be a strong binding of cellsbearing a suitable E-selectin ligand, over a time-course of 20 min to 1hour, particularly during the course of local inflammation.

Phillips et al. (Science 250:1130, 1990) first identified the bindingtarget of E-selectin as the oligosaccharide sialyl Lewis X (sLe^(x))(NeuAcI2,3Galν1,4(fucI1,3)GlcNac-), a terminal structure found on cellsurface glycoprotein of neutrophils. This has become the prototypecarbohydrate ligand for the selectin class. This and relatedoligosaccharides are the subject of U.S. Pat. No. 5,576,305 and PCTapplication WO 92/07572.

The sLe^(x) unit has been assembled into various polymeric structures inan attempt to improve its weak binding to selectins. For example, U.S.Pat. No. 5,470,843 and DeFrees et al. (J. Am. Chem. Soc. 117:66, 1995)disclose bivalent sialyl X saccharides. U.S. Pat. No. 5,470,843discloses a carbohydrate-containing polymer having a synthetic polymerbackbone with 10-20 sLe^(x), sLe^(a), or GlcNac linked via abifunctional spacer.

DeFrees et al. (J. Am. Chem. Soc. 118:6101, 1996) describe a sLe^(x)preparation made with conventional phospholipid liposome technology. Theliposomes contain phosphatidyl choline, cholesterol, phospholipidconjugated with methoxypolyethylene glycol, and phospholipid conjugatedwith sLe^(x) through a polyethylene glycol spacer. Data is presentedshowing that this composition is 5×10³ fold more potent than the sLe^(x)monomer in inhibiting the binding of E-selectin to cells. Murohara etal. (Cardiovasc. Res. 30:965, 1995) tested sLe^(x) phospholiposomes in amyocardial reperfusion model, and found that a dose of 400 Tg/kg bodyweight reduced the proportional size of the area of risk and necrosis.

P-selectin is a transmembrane glycoprotein of ˜140 kDa, substantiallylarger than E-selectin. It was originally described on platelets, inwhich it may be found in I- and dense-granules. Upon activation ofplatelets with a mediator like thrombin, P-selectin is rapidlyredistributed to the cell surface. In endothelial cells, it is found ingranules known as Weibel-Palade bodies, from which it is redistributedto the surface upon activation with histamine. Shuttling of P-selectinto storage granules appears to be mediated by a sorting signal presentin the cytoplasmic domain, and apparently unique in comparison withE-selectin.

Accordingly, P-selectin differs from E-selectin in that it may berapidly expressed from storage granules rather than requiring de novosynthesis. P-selectin binds carbohydrate ligands present on neutrophils,monocytes, and memory T cells. Not only is P-selectin in a preformedstate, its expression is stimulated by mediators such as histamine whichin turn are preformed and stored in the granules of inflammatory cells.The adherence of leukocytes to P-selectin rather than E-selectin onendothelial cells is perhaps the initial event that occurs forrecruitment of these cells to an injured site. Interference withP-selectin binding may be particularly important when it is desirable tolimit leukocyte migration.

The presence of P-selectin on platelets suggests additional uniquebiological roles compared with the other selecting. In one hypothesis,sites of tissue injury may be acutely enriched with short-actingplatelet activators, and active platelets expressing P-selectin maydirectly recruit other leukocytes. In another hypothesis, neutrophils ormonocytes at an inflamed site may be able to catch platelets by way ofthe P-selectin, which in turn could lead to clot formation or additionalmediator release. In an experimental thrombus model, it has beenobserved that platelets accumulate first at the injury site, followed byleukocyte adherence and fibrin deposition. Both of the latter two stepswas inhibited by antibodies against P-selectin (Palabrica et al., Nature359:848, 1992).

L-selectin has a number of features that are different from the otherknown selecting. First, the tissue distribution pattern is opposite tothat of P- and E-selectin--it is expressed on the surface of leukocytes,rather than on the endothelium; while the ligand it binds to is on theendothelium rather than the leukocytes. Second, L-selectin isconstitutively expressed, rather than being up-regulated duringinflammation, and is in fact shed following activation. This may act toallow the activated cells to be released after binding, or may indicatea role of L-selectin in cellular activation. Third, L-selectin ispresent not only on neutrophils and monocytes, but also on mostlymphocytes; while the ligand counterpart is present not only onendothelium but also on lymph node HEV. L-selectin appears to play a keyrole in homing to lymph nodes (Shimizu et al., Immunol. Today 13:106,1992; Picker et al., Annu. Rev. Immunol. 10:561, 1992). In pathologicalconditions involving the immune system, it may be L-selectin that playsthe most central role.

U.S. Pat. No. 5,489,578 describes sulfated ligands for L-selectin andmethods of treating inflammation. The ligands are sulfooligosaccharidesbased on the carbohydrate structures present on the natural L-selectinligand GlyCAM-1.

U.S. Pat. No. 5,486,536 describes the use of sulfatides asanti-inflammatory compounds. The binding activity was attributed to acritical sulfate group at position 3 on the pyranose ring of galactose.In one experiment, sulfatides were sonicated in a protein-containingbuffer to produce microdroplets. The preparation was asserted to haveprotective effects in two animal models for acute lung injury andinflammation.

Each of the selectins shows a fine specificity in terms of thecarbohydrate requirement for binding. All three selectins bindsialylated fucooligosaccharides, of which the prototype is thetetrasaccharide sialyl Lewis^(x) (sLe^(x)). Direct binding experimentsbetween synthetic carbohydrates and isolated selectins has permitted amore detailed dissection of the binding requirements (e.g., Brandley etal., Glycobiology 3:633, 1993). E- and L-selectin require an I2-3linkage for the sialic acid in sLe^(x), whereas P-selectin can recognizesialic acid in an I2-6 linkage. P-selectin also does not require ahydroxyl group in the fucose 2- and 4-positions. P- and L-selectin bindsulfated structures like sulpho-Le^(x) -(Glc)-cer and sulfatides in amanner largely independent of divalent cations, whereas E-selectinbinding is exquisitely sensitive to the presence of cations. Binding ofP- and L-selectin to sulfated carbohydrates is only inhibitible by othersulfated carbohydrates, whereas E-selectin does not have thisrequirement.

It is important to emphasize that the selectin specificity in biologicalreactions is mediated by much more than the carbohydrate component ofthe ligand. For example, P- and L-selectin (but not E-selectin) bindsulfated molecules that lack sialic acid and fucose, such as sulfatides(Aruffo et al., Cell 67:35, 1991) and certain subspecies of heparin(Norgard-Sumnicht et al., Science 261:480, 1993). For a general reviewof the variety of carbohydrates recognized by the selectins, see Varkiet al. (Proc. Natl. Acad. Sci. USA 91:7390, 1994).

Each of the selectins has a different family of natural ligands on thesurface of the opposing cell (see McEver et al., 270:11025, 1995).E-selectin binds strongly to a ligand designated ESL-1. In contrast,antibody blocking studies indicate that essentially all the bindingsites for P-selectin on leukocytes are attributable to an O-glycosylatedprotein designated PSGL-1 (P-selectin glycoprotein ligand 1) (Moore etal., J. Cell Biol. 128:661, 1995). The natural ligands identified forL-selectin is neither of these, but include other glycoproteins with thedesignations GlyCAM-1, CD34, and MAdCAM-1.

The binding specificity indicates that at least two of the threeselectins must be recognizing a ligand component beyond the sLe^(x)structure. In addition to the oligosaccharide, P-selectin must bind asite on PSGL-1 with features different from ESL-1 and from othermucin-like O-glycosylated proteins, such as CD43.

A second ligand requirement for high affinity binding of the naturalligand has been identified for both P- and L-selectin. The secondrequirement is a sulfate residue, which is apparently not required forE-selectin binding, and has implications for the development ofeffective inhibitory compounds.

Imai et al. (Nature 361:555, 1993) tested the requirements for bindingof L-selectin to the ligands on lymph node HEV. Radioactive inorganicsulfate is incorporated into the 50 kDa and 90 kDa glycoproteins in amanner that is inhibitible by sodium chlorate. The undersulfatedglycoproteins no longer interacted in precipitation analyses with anL-selectin chimera. The inhibition experiments do not pinpoint thelocation of the required sulfate group to the carbohydrate or theprotein backbone. Either way, the sulfate requirement distinguishesL-selectin binding specificity from that of E-selectin.

The sulfate component has been mapped more precisely in the structure ofthe P-selectin ligand PSGL-1. The requirement in P-selectin is providedby one or more sulfated tyrosines near the N-terminus of the polypeptidebackbone, separate from the glycosylation site.

Wilkins et al. (J. Biol. Chem. 270:22677, 1995) demonstrated that PSGL-1synthesized in human HL-60 cells can be metabolically labeled with [³⁵S]sulfate. It was shown that most of the ³⁵ S label was incorporatedinto the polypeptide in the form of tyrosine sulfate. Treatment ofPSGL-1 with a bacterial arylsulfatase released sulfate from tyrosine,and resulted in a concordant decrease in binding to P-selectin.

Pouyani et al. (Cell 83:333, 1995) demonstrated that selectiveinhibitors of sulfation compromised binding of HL-60 cells to solubleP-selectin but not E-selectin. The cell-surface expression of sLe^(x) orthe polypeptide were not compromised by treatment. Deletion analysis ofisolated PSGL-1 constructs localized the binding component to residues20-40. The segment contains three tyrosine residues, and when these werechanged to phenylalanine, P-selectin binding activity was abolished.Furthermore, when the 20 amino acid segment was fused on to a differentprotein, it was again sulfated during biosynthesis and had bindingactivity for P-selectin. These authors suggested that the sulfatedtyrosines interact with P-selectin not through the carbohydrate bindingdomain of P-selectin, but through the EGF-like domain, which is locatedcloser in the protein sequence to the membrane spanning domain.

Sako et al. (Cell 83:323, 1995) performed another series of bindingexperiments using the extracellular domain of PSGL-1 expressed as afusion protein. The assay required fucosylation of the protein andcations in the assay medium, consistent with a dependence oncarbohydrates like sLe^(x). Mutation of the putative N-linkedglycosylation sites had no effect on selectin binding, suggesting thatthe carbohydrate requirement was O-linked. However, mutation of threetyrosines to phenylalanine abrogated binding activity for P-selectin.Binding of E-selectin, for which PSGL-1 can also act as a ligand, wasnot affected by removal of the sulfation sites.

The binding affinity of P- and L-selectin for sLe^(x) is in the mM range(Nelson et al., J. Clin Invest. 91:1157, 1993). In contrast, theaffinity of P-selectin for the natural ligand is in the nM range (Mooreet al., J. Cell Biol. 112:491, 1991), a difference in potency of ˜10⁶fold. Synthetic oligosaccharides containing multiple sLe^(x) units onlypartly make up the difference, so the effect is not just due to ligandvalency. The disparity is also attributable to the requirement of P- andL-selectin for a strong anionic determinant, like the sulfotyrosines on

PSGL-1. Compounds effective in the same concentration range as PSGL-1must be able to supply a similarly effective determinant combination.

There is a need to develop new therapeutic compositions capable ofinterfering with selectin-ligand interactions, because cellular adhesionis an early event in a number of inflammatory and immunologicalphenomena. For systemic administration, the compositions should beeffective in the nanomolar range, so that an effective amount can begiven in a practicable dose. It is important to emphasize that putativecompositions should be tested in a system that adequately represents therequirements of the natural interaction. A one-component inhibitor thateffectively blocks a one-component interaction will typically not beeffective in blocking a two-component interaction.

This disclosure describes polymerized lipid compositions that displayall the features necessary to inhibit P- or L-selectin at nanomolarconcentrations when tested in appropriate cell bioassays for ligandbinding. Polymerized liposomes and lipid sheets have been proposed inother contexts (Spevak et al., Adv. Mater 7:85, 1995; Reichert et al.,J. Am. Chem Soc. 117:829, 1995; Charych et al., Science 261:585, 1993;Charych et al., Chem. Biol. 3:113, 1996). However, the present inventionis the first instance where polymerized glycoliposomes have been shownto be effective in a biological system involving the interaction of twoeukaryotic cells. This is also the first instance where polymerizedglycoliposomes have been shown to be an effective ligand for a bindingsystem with a plurality of separate determinants.

SUMMARY OF THE INVENTION

The lipid compositions of this invention provide a stable scaffold fromwhich to present a plurality of features required for ligand binding. P-and L-selectin inhibitors comprise a multivalent assembly ofcarbohydrates, interspersed with negatively charged lipid headgroupswhich are essential for activity. These compositions are proposed foruse in inhibiting biological phenomena mediated by selecting, includingthe adherence and extravasation of neutrophils and monocytes, and thetrafficking of lymphocytes through blood vessels, lymphatics, anddiseased tissue.

Accordingly, certain embodiments of this invention relate tocompositions for inhibiting the binding between a first cell having a P-or L-selectin and a second cell having a ligand for the selectin,comprising a sheet of lipids wherein a proportion of the lipids arecovalently crosslinked, a proportion of the lipids have an attachedsaccharide, and a proportion of the lipids not having an attachedsaccharide have an acid group that is negatively charged at neutral pH.A proportion of the lipids having the attached saccharide or the acidgroup may be covalently crosslinked to other lipids in the sheet, and aproportion may not be ocvalently crosslinked to other lipids.

This includes embodiments wherein a proportion of the lipids in thelipid sheet have a first attached saccharide, and a separate proportionof the lipids in the lipid sheet have a second attached saccharide thatis different from the first. The composition preferably has a 50%inhibition concentration (IC₅₀) that is 10² -fold or 10⁴ -fold lowerthan that of monomer sLe^(x).

Also embodied in this invention are compositions for inhibitingleukocyte adhesion or migration; compositions for inhibiting leukocyteadherence or fibrin deposition; compositions for inhibiting leukocyteadhesion or migration, compositions for inhibiting lymphocyte adhesion,and compositions for other types of interventions in cell interactionmediated by selectin, comprising inhibiting binding between a first cellhaving a P- or L-selectin and a second cell having a ligand for theselectin as already outlined

Another embodiment of the invention is a composition for inhibiting thebinding between a P- or L-selectin and a ligand for the selectin,wherein the lipid composition containing the ligands comprises a sheetof lipids wherein a proportion of the lipids are covalently crosslinked,a proportion of the lipids have an attached saccharide, and a proportionof the lipids not having an attached saccharide have an acid group thatis negatively charged at neutral pH.

Also embodied is a composition for selecting polymerized glycoliposornewith selectin binding activity, comprising the steps of providing aglycoliposome with covalently crosslinked lipids, and a saccharideattached to a proportion of the covalently crosslinked lipids;introducing the glycoliposome into an environment comprising a selectinand a cell having a selectin ligand; and selecting the glycoliposome ifthe relative inhibitory concentration is lower than that of monomersLe^(x).

Also embodied is composition comprising a polymerized lipid compositionin the manufacture of a medicament for use in treating a diseasecharacterized by local alteration in the adherence of leukocytes orcancer cells to vascular endothelium, platelets or lymphatic tissue;particularly diseases of inflammatory or immanological etiology; whereinthepolymerized lipid composition comprises a sheet of lipids wherein aproportion of the lipids are covalently crosslinked, a proportion of thelipids have an attached saccharide, and a proportion of the lipids nothaving an attached saccharide have an acid group that is negativelycharged at neutral pH.

Also embodied are compositions for treating a disease characterized bylocal alteration in the adherence of leukocytes or cancer cells tovascular endothelium, platelets or lymphatic tissue, conprising apolymerized lipid composition comprising a sheet of lipids wherein aproportion of the lipids are covalently crosslinked, a proportion of thelipids have an attached saccharide, and a proportion of the lipids nothaving an attached saccharide have an acid group that is negativelycharged at neutral pH. Diseases of interest include but are not limitedto cardiac disease (such as ischernia reperfusion injury, myocardialinfarction, myocarditis, restenosis, and deep vein thrornbosis),hemmorhagic shock, arthritis, asthma, and metastatic cancer.

Also embodied are compositions with P- and L-selectin inhibitoryactivity and pharmaceutical compositions prepared therefrom, as may berecited in any of the aforementioned methods or described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing of two polymerized glycoliposomes showing anexpanded detail of the chemical structure. Structure "A" is able toinhibit the binding of P-selectin to HL-60 cells at an oligosaccharideconcentration below 2 nM, while Structure "B" has essentially noactivity. The vesicles are unilamellar and made up of single-chainlipids with diyne groups cross-linked using UV light. Conjugated toabout 5% of the lipids are analogs of the sLe^(x) oligosaccharide. Thepreparations differ in terms of the outward facing determinantsdisplayed by the neighboring lipids. In structure "A", the neighboringlipids provide carboxylic acid groups, which have a negative charge atneutral pH. In structure "B", the neighboring lipids are neutral. Thenegatively charged lipids work synergistically with the sLe^(x) analogto supply P-selectin binding activity, just as sulfotyrosine workssynergistically with sLe^(x) in the natural ligand. P- and L-selectindiffer from E-selectin in the requirement for a negative chargedeterminant in binding.

FIGS. 2A and 2B depict some of the aspects of selectin binding. In FIG.2A the boxed panel shows the receptor ligand pairs known for L-, P- andE-selectin. They are depicted on the same cell for convenience, butparticipate in different ways to cell adhesion and migration. FIG. 2B isa detail showing the dual binding site model for P-selectin. In theligand PSGL-1, the negative groups correspond to three sulfotyrosineresidues. In contrast, there is no evidence for a separate anion bindingsite for E-selectin.

FIG. 3 is a drawing of particular components that may be chosen forassembly into glycoliposomes of this invention.

FIG. 4 is a titration curve for the inhibition of P-selectin binding toHL-60 cells by glycoliposomes. In order of decreasing potency (left toright) the compositions are comprised of: sLe^(x) analog plus acidiclipids; lactose plus acidic lipids; maltose plus acidic lipids; andsLe^(x) analog plus neutral lipids.

FIGS. 5A and 5B are bar graphs showing the 50% inhibition concentrationof various glycoliposome preparations.

FIGS. 6A through 6E are drawings of polymerized liposomes tested forbinding in Example 3. Amongst the components tested, the sulfo Le^(x)analog was found to be the best carbohydrate, and lipid with a sulfategroup best fulfilled the requirement for a separate negatively chargedgroup.

FIGS. 7 and 8 are drawings of additional exemplary carbohydratedeterminants for inclusion in polymerized glycoliposomes.

FIG. 9 is a drawing comparing the sLe^(x) structure and an sLe^(x)tethered analog with a novel glycoliposome comprising sialic acid andfucose residues on neighboring lipids in the crosslinked matrix.

DETAILED DESCRIPTION

It is an object of this invention to provide a system for inhibition ofthe binding of P- and L-selectin to their counterpart ligands,especially during the interaction between two cells. Polymerized lipidcompositions are contacted with one of the interacting cells, or elseintroduced into an environment where the cells are expected to interact.This type of intervention is of therapeutic interest in any circumstancewhere the adherence, migration, or activation of cells is mediated by aselectin, and adverse to the well-being of the host.

Polymerized glycolipid compositions for use in this invention minimallycomprise three elements:

1. A stable platform made up of a lipid sheet stabilized by covalentcrosslinking between a proportion of the lipids.

2. A saccharide or similar structure attached to a lipid in the lipidsheet that meets the carbohydrate binding requirement of selecting.Typically, the glycolipid is one of the crosslinked lipids in thestructure, but it may instead be trapped between other lipids that formthe crosslinked scaffold.

3. A negatively charged or electronegative group (usually a carboxylicacid or oxyacid) that meets the anionic binding requirement of P- andL-selectin. There is no requirement that the group play exactly the samerole as the sulfotyrosines of PSGL-1, as long as the anionic bindingrequirement is satisfied.

When exemplary compositions were prepared and tested for inhibitoryactivity in a cell bioassay, a number of important observations weremade that underscore the improvement provided by this technology.

Polymerized liposomes have not been tested previously for inhibition ofmulti-component binding. The relative positioning of the saccharide andthe negatively charged group is a chance of random polymerization, not acontrolled structure as it is in stepwise chemical synthesis of smallmolecules. It could not be predicted that an effective orientation wouldresult, but it was found that active compositions are reproduciblyproduced without difficulty. New determinant combinations are easilyassembled and tested for activity.

The negatively charged group of the natural ligand PSGL-1 issulfotyrosine, and the nature of what would be required to satisfy theanionic binding requirement in liposomes was unknown. It was found thatthe anionic binding requirement does not require the anion to be on aprotein or carbohydrate component, but can be directly coupled to lipidsthat become part of the lipid sheet. Surprisingly, the anionic componentneed not be a sulfate group, but can be provided as a simple carboxylicacid headgroup on the lipid.

The presence of the acid group on neighboring lipids unexpectedlyreduced the stringency of the oligosaccharide requirement. Neutraldisaccharides such as lactose and maltose have not previously been shownto have any selectin binding activity, and were included in the initialexperiments as "negative controls". Unexpectedly, compositionscontaining these sugars and anionic lipids were potent selectininhibitors. This is of considerable commercial interest, because themanufacture of compositions containing sugars like lactose is easier andless expensive than those containing more complex sugars such assLe^(x).

The inhibitory activity was remarkably high. In the cell bioassay, thesLe^(x) analoganionic lipid combination had an IC₅₀ as low as 2 nM,which is up to 10⁶ -fold lower than sLe^(x) monomer. The lactose anioniclipid combination was effective at 15 nM. This means that an effectivetherapeutic dose can be prepared at a lower cost and administered in asmaller volume than prior art compositions.

FIG. 1 shows exemplary lipid compositions of this invention, in which ananalog of sLe^(x) is displayed on the surface of a polymerizedunilamellar liposome. Only the first structure demonstrated inhibitoryactivity for P-selectin binding in the bioassay, underlining theimportance of the anionic component in the composition.

Because the carbohydrate and anionic determinants are on separate lipidsin the polymerized lipid compositions, another benefit of the approachdescribed here is that the components can be separately screened andtitrated to produce improved compositions with refined bindingcharacteristics.

Preparation of Polymerized Lipid Compositions

It will be readily appreciated from the drawing in FIG. 1 and the dataprovided in Example 2 that the practice of this invention is notcritically dependent on the chemical details of the composition. Withinthe constraints of the three requirements above, the practitioner isfree to assemble the composition according to a number of differentapproaches. Variations in polymerization chemistry and the conjugationof determinants are permitted and included in the scope of thisinvention. Designing particular linkages between a carbohydrate and alipid is well within the skill of the ordinary practitioner. Theoptimization of the compounds may achieved by routine adjustment andfollowing the effects of adjustment on selectin binding in one of manyassays established in the art.

The following section is provided merely as an illustration of possibleapproaches for the convenience of the reader.

Preparation of components of the lipid composition: The invention useslipids both to bear the determinants required to inhibit selectinbinding, and as components for forming the lipid assemblies. Examples oflipids that can be used in the invention are fatty acids, preferablycontaining from about 8 to 30 carbon atoms in a saturated,monounsaturated, or multiply unsaturated form; acylated derivatives ofpolyamino, polyhydroxy, or mixed aminohydroxy compounds;glycosylacylglycerols; phospholipids; phosphoglycerides; sphingolipids(including sphingomyelins and glycosphingolipids); steroids such ascholesterol; terpenes; prostaglandins; and non-saponifiable lipids.

The negatively charged group of the composition is typically an acidaccessible from the exterior surface of the lipid sheet. In certainembodiments, the acid is an organic acid, particularly a carboxylicacid. In other embodiments, the acid is an oxyacid of the form(XO_(n))(O⁻)_(p), wherein n+p>2. In this case, the lipid will typicallybe of the form R_(m) (XO_(n))(O⁻)_(p) wherein each R comprises analiphatic hydrocarbon (which are not necessarily the same), m is 1 or 2,(XO_(n))(O⁻)_(p) is an oxyacid, and n+p>2. Preferred oxyacids aresulfate, SO₃ ⁻, and phosphate. A phosphate may be conjugated through oneor two of its oxygens to aliphatic hydrocarbons. For any negativelycharged component of the composition, any additional features may bepresent between the acid and the aliphatic or membrane anchoring group.These include spacers such as polyethylene glycols and otherheteroatom-containing hydrocarbons. The acid group may also be presenton a substituent such as an amino acid, a sugar, or a pseudo-sugar,which includes phosphorylated or sulfated forms of cyclohexidine,particularly hexaphosphatidyl inositol and hexasulfatidyl inositol.

The negatively charged group may already be present in the lipid, or maybe introduced by synthesis. Examples of lipids with negatively chargedheadgroups include the fatty acids themselves (where the negative chargeis provided by a carboxylate group), cardiolipin (phosphate groups),dioleoylphosphatidic acid (phosphate groups), and the 1,4-dihexadecylester of sulfosuccinic acid (sulfate group).

Negatively charged lipids not commercially available can be synthesizedby standard techniques. A few non-limiting illustrations follow. In oneapproach, fatty acids are activated with N-hydroxysuccinimide (NHS) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in methylenechloride. The leaving group N-hydroxysuccinimide can be displaced with awide range of nucleophiles. In one example, glycine is used to yield afatty acid-amino acid conjugate with a negatively charged headgroup.Glutamic acid can be coupled to the activated fatty acid to yield afatty acid-amino acid conjugate with two negative charges in itsheadgroup. In another synthetic approach,2,3-bis((1-oxotetradecyl)oxy)-butanedioic acid is prepared by addingmyristoyl chloride in toluene to a pyridine solution of di-tartaricacid. The clarified solution is concentrated to yield the product, whichis recrystallized from hexane (Kunitake et al., Bull. Chem. Soc. Japan,51:1877, 1978).

A sulfated lipid, the 1,4-dihexadecyl ester of sulfosuccinic acid, isprepared as follows: a mixture of maleic anhydride and hexadecyl alcoholin toluene with a few drops of concentrated sulfuric acid is heated withazeotropic removal of water for 3 h. The dihexadecyl maleate isrecrystallized, then heated with an equimolar amount of NaHSO₃ in waterat 100° C. for 2-3 h. The product is recovered by evaporating the waterand extracting the lipid into methanol (Kunitake et al., supra). Alkylsulfonates may be synthesized as follows. A lipid alcohol is obtainedfrom Sigma, or the acid group of a fatty acid is reduced to an alcoholby reacting with lithium aluminum hydride in ether to convert thecarboxylate into an alcohol. The alcohol can be converted into a bromideby reaction with triphenylphosphine and carbon tetrabromide in methylenechloride. The bromide is then reacted with bisulfite ion to yield thealkyl sulfonate. Sulfates may be prepared by reacting an activated fattyacid with a sulfate-containing amine. For example, theN-hydroxysuccinimide ester of 10, 12-pentacosadiynoic acid is reactedwith taurine to yield N-10, 12-pentacosadiynoyl taurine. Sulfates mayalso be prepared by reacting an alcohol, e.g. lauryl alcohol, withsulfur trioxide-trimethylamine complex in anhydrous dimethylformamidefor 2.5 h (Bertozzi et al., Biochemistry 34:14271, 1995).

Phosphate-containing lipids not commercially obtainable are also readilysynthesized. For example, to prepare dialkyl phosphate compounds,phosphoryl chloride is reacted with the corresponding alcohol. To makedihexadecyl phosphate, phosphoryl chloride is refluxed with threeequivalents of hexadecyl alcohol in benzene for twenty hours, followedby recrystallization of the product (Kunitake et al., supra). Monoalkylphosphates may be prepared by reacting, e.g., 10, 12-hexacosadjyne-1-ol(1 eq.) with phosphoryl chloride (1.5 eq.) at ambient temperature in dryCCl₄ for ˜12 h, then boiling under reflux for 6 h. Removal of thesolvent and heating the residue with water for 1 h yields the desired10, 12-hexacosadiyne-1-phosphate (Hupfer et al., Chem. Phys. Lipids33:355, 1983). Alternatively, a fatty acid activated with NHS can bereacted with 2-aminoethylphosphate to yield the acylated derivative ofaminoethylphosphate.

Carbohydrate components suitable for use with this invention include anymonosaccharides, disaccharides, and larger oligosaccharides withselectin binding activity when incorporated into a polymerized lipidsheet. Simple disaccharides like lactose and maltose have no selectinbinding activity as monomers, but when incorporated into polymerizedliposomes acquire substantial activity. Accordingly, the range ofsuitable carbohydrates extends considerably beyond what is used in otherselectin inhibitors.

In some embodiments, the carbohydrate is a disaccharide or neutralsaccharide with no detectable binding as an unconjugated monomer. Inother embodiments, the carbohydrates have substantial binding in themonomeric form, and are optionally synthesized as a multimericoligosaccharide, although this is not typically required. Preferredoligosaccharides are sialylated fucooligosaccharides, particularlysLe^(x) and sLe^(a), analogs of sialylated fucooligosaccharides,sulfated fucooligosaccharide, particularly sulfo Le^(x), and analogs ofsulfated fucooligosaccharide. Disaccharides and larger oligosaccharidemay optionally comprise other features or spacer groups of anon-carbohydrate nature between saccharide units.

A "sialylated fucooligosaccharide analog" is a saccharide that containsthe minimal structural components of sLe^(x) involved in selectinbinding in a spatially similar orientation to that of sLe^(x). Thesecomponents are the 3-hydroxy group of the fucose subunit and thenegatively group of the neuraminic acid subunit of sLe^(x). In thecontext of L-selectin binding, preferred analogs include the 2-, 3-, and4-hydroxy groups of the fucose subunit and the negatively charged groupof the neuraminic acid subunit. The fucose and sialic acid componentsmay be linked through a disaccharide spacer as they are in sLe^(x),through a hydrocarbon linker (as in the tethered analogs exemplifiedbelow), or through a synthetic spacer of appropriate length containingsuch optional features as cyclic and aromatic groups. Exemplars of thelatter type are listed in the review by Sears et al. (Proc. Natl. Acad.Sci. USA 93:12086, 1996)--see especially FIG. 7.

Certain analogs and other oligosaccharides of particular interestinclude the following: 1. Tethered disaccharides, containing a spacerbetween two sugars, particularly sialic acid or a sulfated form thereofand fucose, wherein the spacer is a linear or branched alkyl group (FIG.9) or mixed hydrocarbon (Hanessian et al., J. Syn. Lett. 868, 1994). 2.Analogs comprising a fucose residue and the carboxylic acid group ofsialic acid connected by hydroxylated ring structures (Lin et al.,Biorganic Med. Chem. Lett 6:2755, 1996). 3. Lactose sulfated at one ormore positions (Bertozzi et al., Biochemmistry 34, 14271, 1995). 4.Neutral disaccharides with an ether linkage to a carboxylic acid group(Hiruma et al., J. Am. Chem Soc. 118:9265, 1996). 5. A monosaccharide(not necessarily fucose) linked through multiple 5- or 6-member ringstructures to a carboxylic acid group, at least one of the ringstructures being a phenyl group (Dupre et al., Bioorg. Med. Chem. Lett.,6:569, 1996). 7. Glycopeptides, comprising a fucose or similarmonosaccharide linked via a plurality of peptide bonds to a carboxylicacid (Cappi et al., Angew. Chem. Int. Ed. Engl. 1996; Wang et al.,Tetrahedron Lett. 37:5427, 1996). 8. Tri- and tetrasaccharides with aplurality of sulfate groups (Nelson et al., Blood 82:3253, 1993). 9.Phosphorylated or hydroxylated cyclohexanes, particularlyhexaphosphatidyl inositol and hexasulfatidyl inositol (Cacconi et al.,J. Biol. Chem. 269:15060, 1994).

Many mono and disaccharides are available commercially. The syntheses ofmore complex carbohydrate structures for selectin binding are describedextensively in the art, and need not be elaborated here. Academicarticles of interest to the reader may include Tonne et al. (Tetrahedron45:5365, 1989); Drueckhammer et al. (Synthesis 499, 1989); Hindsgaul(Sem. Cell Biol. 2:319, 1991); Look et al. (Anal. Biochem. 202:215,1992); Ito et al. (Pure Appl. Chem. 65:753, 1993); and DeFrees et al.(J. Am. Chem. Soc. 117:66, 1995).

Conjugation of carbohydrates onto lipids can be conducted by anyestablished or devised synthetic strategy, suitably protecting thecarbohydrate during conjugation as required. One method is to react afatty acid activated by N-hydroxysuccinimide with an amino sugar such asglucosamine or galactosamine. If an oligosaccharide-lipid conjugate isdesired, the oligosaccharide may be synthesized first, utilizing anamino sugar as one of the subunits. The amino group of the amino sugaris then acylated by the activated fatty acid to yield thelipid-oligosaccharide conjugate. It should be noted that in anoligosaccharide, the amino sugar-fatty acid conjugation may interferesterically with binding to the desired target. Thus it may be desirableto extend the oligosaccharide by interposition of other sugar subunitsbetween the amino sugar-lipid conjugate and the portion of thesaccharide acting as a ligand. For example, for sLe^(x), the aminosugar-fatty acid conjugation may introduce steric hindrance of bindingif the amino sugar is too close to the binding moieties of the sLe^(x).Thus the sLe^(x) should be extended by coupling the amino sugar to theGlcNAc subunit of sLe^(x) via an O-glycosidic bond, instead ofsubstituting the amino sugar for the GlcNAc subunit, in order to avoidsteric hindrance of binding.

Another method utilizing the amino group of an amino sugar is tointroduce an iodoacetyl group onto the amino group, followed by reactionof the amino group with a thiol-containing compound (such as cystamineor cysteine) which contains additional functional groups for furtherderivatization.

O-glycosides are readily formed by the acid-catalyzed condensation of analcohol with monosaccharides such as glucose or mannose.N-Fmoc-ethanolamine can be added to the reducing end of glucose,followed by deprotection of the amino group with piperidine. The freeamino group of the compound can then be acylated with an activated fattyacid to form a carbohydrate-lipid conjugate. Alternatively, glycosylhalides (formed by reacting a sugar with a haloacid such as HCl) can beused, where nucleophilic displacement of the halide by an alcohol formsthe O-glycoside.

Another method involves the formation of N-glycosides by reacting anamine with a reducing sugar. This reaction is readily accomplished byreacting the sugar, e.g. glucose, with an amine, e.g. decylamine, atambient temperature for ˜48 h. Alternatively, heating the sugar with theamine, e.g. stearylamine (in 2-3 molar excess) at 80° C. in anethanol/water solution will suffice to form the N-stearyl glycoside(Lockhoff, Angew. Chem. Int. Ed. Eng. 30:1161, 1991). In order toincrease the stability of the N-glycoside, the product is peracetylatedby stirring in 60% pyridine/40% acetic anhydride at 0° C. Theperacetylated product is then dissolved in anhydrous methanol, 1M sodiummethoxide is added to adjust the pH to ˜10), and the mixture stirred atroom temperature for 3 h to yield the N-acetyl-N-glycoside.

An extension of this method of introducing additional functionality viaN-glycosides involves the addition of a polyfunctional amine to thesugar. For example, N-allylamine can be added to a saccharide with afree reducing end, followed by reaction of the allyl group to provide asuitable point of attachment for a fatty acid. One of skill in the artwill recognize that the sugar conjugates depicted in FIG. 3 are createdby reacting N-allylamine with sLe^(x) analog, followed by peracetylationof the N-glycoside. The hydroxyl groups can be deprotected with acatalytic amount of sodium methoxide, resulting in the N-acetylatedN-allyl glycoside. Alternatively, the amino group of the N-allylglycoside can be directly acetylated with an acid chloride (Lockhoff,Angew. Chem. Int. Ed. Eng. 30:1161, 1991). A mercaptoamine such ascystamine can then be added to the N-allyl glycoside by irradiation withUV light (Roy et al., J. Chem. Soc. Chem. Comm. 1059, 1988), whichresults in an N-glycoside with a free amino group. The free amino groupcan then be readily coupled to an activated fatty acid such as theN-hydroxysuccinimide ester of 10, 12-pentacosadiynoic acid to yield theconjugated sugar.

Other methods of attaching fatty acids or other lipids to carbohydratescan be accomplished by forming suitable thioglycosides or C-glycosides.These compounds can then be further derivatized in a manner analogous tothe methods used for the N-glycosides. The C-allyl glycoside ofneuraminic acid, for example, is readily formed by reaction of N-acetylmannosamine and sodium pyruvate in the presence of NeuAc aldolase ascatalyst to yield N-acetyl neuraminic acid. Treatment of the crudereaction mixture with HCl gas in ethanol yields an ethyl ester; this isfollowed by reaction with acetyl chloride to give a glycosyl chloride(this step also results in acetylation of the hydroxyl groups). Reactionof this glycosyl chloride with allyl tributyltin and a catalytic amountof bis (tributyltin) under UV irradiation (a 450 Watt Hanovia lamp,equipped with a Pyrex filter) yields a C-allyl glycoside; the acetylgroups are then removed from the hydroxyl groups with sodium ethoxide inethanol. This yields the ethyl ester of the C-allyl glycoside ofneuraminic acid (Nagy, J. O. et al., Tetrahedron Letters 32:3953 1991).

In a manner analogous to the reaction scheme described above for theN-allyl glycosides, the C-allyl glycoside of a sugar may be reacted withcystamine, resulting in the addition of the thiol group to the allylgroup, followed by reaction of the amino group with an activated fattyacid.

Conjugation of a carbohydrate to a lipid via an amide bond may beaccomplished if the carbohydrate has a free carboxyl group. Mixing thecarbohydrate and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-ethanamine andactivating the carboxyl group by using1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and1-hydroxybenzotriazole (HOBt) in methylene chloride, followed byreduction of the azido group to an amine with H₂ /Pd(OH)₂ /C inethanol/water/dioxane/acetic acid (2:1:2:1), yields an amine-derivatizedcarbohydrate which can then be linked to a fatty acid by a variety ofactivating chemistries (Lin et al., Bioorg. & Med. Chem. Lett. 6:2755,1996).

Carbohydrates can also be conjugated to lipids using enzymatic methods.Sugars may be transphosphatidylated by reacting diacylphosphatidylcholine and the sugar in the presence of phospholipase D, resulting inthe diacylphosphatidyl-sugar (Wang et al., J. Am. Chem. Soc. 115:10487,1993).

Assembly of the lipid composition: Appropriately derivitized lipids arecombined, formed into a suitable composition, and cross-linked.

Where appropriate, the combination step includes mixing lipids havingthe carbohydrate requirement for selectin binding with lipids having theanionic requirement. Additional lipids can also be included for avariety of purposes. The additional lipids may have a differentcarbohydrate, or they may be scaffold lipids that participate incrosslinking but have no binding determinant, or they may be fillerlipids that do not have crosslinking groups. Non crosslinked lipids maybear either the carbohydrate determinant, or the anionic determinant, orboth, and become stabilized in the composition by entrapment betweenother crosslinked lipids.

The lipids are then formed into a lipid composition. Although the lipidcompositions are most typically liposomes, any other arrangement can beused providing it is deliverable to the intended site of action, anddisplays the determinants needed for selectin binding. The participatinglipids are crosslinked members of a lipid sheet, but the lipid sheetneed not be part of a lipid bilayer. Micelles and microdroplets areexamples of alternative particulate forms suitable for displaying thebinding determinants. A single lipid sheet may also be formed about ahydrophobic core of a suitable aliphatic compound. Lipid can also beseeded as a single sheet or bilayer about another core substance, suchas a protein complex. Any descriptions in this disclosure that refer toliposomes also apply to other types of lipid compositions, unlessrequired otherwise.

Liposomes are the more usual form of the composition, because of theirease of manufacture. A number of methods are available in the art forpreparing liposomes. The reader is referred to Gregoriadis (ed):"Liposome technology 2nd ed. Vol. I Liposome preparation and relatedtechniques", CRC Press, Boca Raton, 1993; Watwe et al. (Curr. Sci.68:715, 1995), Vemuri et al. (Pharm. Acta Helvetiae 70:95 1995), andU.S. Pat. Nos. 4,737,323; 5,008,050; and 5,252,348. Frequently usedtechniques include hydration of a lipid film, injection, sonication anddetergent dialysis. When using diyne chemistry and single-chain fattyacids for crosslinking, a preferred method is sonication, as describedin one of the original articles (Hub et al., Angew. Chem. Int. Ed. Engl.19:938, 1980). This method is easy to use and produces unilamellarspherical vesicles of small and uniform size. Briefly, a thin film oflipid is heated with water above 90° C., and then cooled to about 4° C.,which is below the T_(c) (Lopez et al., Biochim. Biophys. Acta 693:437,1982) to permit the lipids to form a "solid analogous" state. Themixture is then sonicated for several minutes, with longer times (˜15min) typically producing more uniform vesicles.

After formation, the vesicles may be reduced in size, if desired, byfreeze-thaw cycles or extruding through filters of progressively smallerpore size. Vesicles of any diameter are included within the scope ofthis invention, but they are preferably less than about 400 nm in mediandiameter, and more preferably less than about 200 nm in diameter.Smaller sized vesicles can be sterile-filtered and are less susceptibleto uptake by phagocytic cells.

The lipids used in any of these compositions will have been preparedwith fictional groups that can be covalently crosslinked once the lipidsheet is formed.

Several approaches are known in the art for covalently crosslinkinglipids Polymerization may be accomplished by irradiation withultraviolet light, or by radical initiation with compounds such ashydrogen or benzoyl peroxide, as appropriate, of lipid diynes,styrene-containing lipids, acrylic-containing lipids, and lipid dienes;polymerization (by forming amide bonds) of lipids containing free(unprotected) amino and carboxyl groups; and polymerization (byoxidation of thiol groups) of thiol-containing lipids (wherein eachlipid must contain at least two thiol groups in order to becrosslinked). Azides, epoxides, isocyanates and isothiocyanates, andbenzophenones also afford methods of crosslinking lipids (Wong, S. S.,Chemistry of Protein Conjugation and Cross-Linking, Boston: CRC Press,1993; Hermanson, G. T., Bioconjugate Techniques, San Diego: AcademicPress, 1996).

An example of polymerization of lipids by forming amide bonds is thepolymerization ofN-ε-palmitoyl-L-lysine-N-β-(2-acetylamino-2-deoxy-β-glucopyranosyl)-L-asparagineby carbodiimides. The carbohydrate, lipid-modified dipeptide is readilyassembled by standard solid phase peptide synthesis methods usingcommercially availableN-α-Fmoc-N-β-(3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-glucopyranosyl)-L-sparagine(from Novabiochem) and N-α-Fmoc-N-ε-palmitoyl-L-lysine (which is readilysynthesized by coupling palmitic acid activated withN-hydroxysuccinimide to the free ε-amino of commercially availableN-α-Fmoc-L-lysine). Removal of the modified dipeptide from thesolid-phase resin and deprotection of the fuinctional groups is carriedout by standard methods. The carbohydrate, lipid-modified dipeptide canbe co-polymerized with a second dipeptide,N-ε-palmitoyl-L-lysine-L-aspartic acid, in order to provide a liposomewith both carbohydrate-bearing and negatively-charged groups on itssurface.

An example of polymerization of lipids by oxidation of thiol groups isas follows: 10-undecenoic acid (10-undecylenic acid) is brominated byaddition of HBr by Markonikov addition across the double bond, resultingin 10-bromoundecanoic acid (Streitweiser et al., Introduction to OrganicChemistry, New York: Macmillan, 1976, pp. 278-285). 10-thioundecanoicacid is prepared by treatment of 10-bromoundecanoic acid with thioureain ethanol and subsequent hydrolysis by aqueous NaOH. (Streitweiser etal., Introduction to Organic Chemistry, New York: Macmillan, 1976, pp.242-243). The thiol is then protected with the trityl group by heatingwith triphenylmethanol and boron trifluoride etherate in glacial aceticacid, followed by workup with ethanol, water, and powdered sodiumacetate (Bodanszky et al., The Practice of Peptide Synthesis, New York:Springer-Verlag, 1984, p. 83). The protected thiol fatty acid is thenactivated with N-hydroxysuccinimide and reacted with S-trityl-L-cysteine(Novabiochem). The fatty acid-amino acid conjugate is then treated withtrifluoroacetic acid to remove the trityl groups, resulting inN-(10'-thioundecanoyl)-cysteine. The dithiol can then be polymerized byoxidation with molecular oxygen.

Additional examples of lipids that can be crosslinked are reviewed inRingsdorf et al., Angew. Chemie Int. Ed. Eng., 27:113-158 (1988), andreferences therein, and Johnston, D.S. et al., "Polymerized Liposomesand Vesicles," Chapter 9 in Liposome Technology, Vol. 1 (G. Gregoriadis,Ed.), Boca Raton, Fla.: CRC Press, 1984, pp. 123-129 and referencestherein.

A preferred method of polymerizing lipids is by polymerization of lipiddiynes such as 10, 12-pentacosadiynoic acid (Farchan Laboratories,Gainesville, Fla.) by ultraviolet light. Polymerization reactions ofdiacetylenic compounds have been extensively studied and have beenutilized in the formation of polymerized liposomes, micelles, and othersupramolecular assemblies (see Frankel et al. J. Am. Chem. Soc.113:7436, 1991; Furhop et al., J. Am. Chem. Soc. 113:7437-7439, 1991;Spevak et al., Advanced Materials 7:85, 1995). Diynes are convenientbecause they are easily polymerized using U.V. light, obviating the needfor a radical initiator. In addition, the polymerized lipid is coloredand the degree of polymerization can be easily monitored.

An example of the preparation of a crosslinkable diacyl lipid, 1, 2,3-triamino-(bis-N1, N3-pentacosa-10, 12-diynoyl) propane, is as follows.The t-butyloxycarbonyl (Boc) group is used to protect the amino group of2-amino-1,3-propanediol. The diol is converted into a di-mesylate withmesyl chloride, followed by immediate reaction with tetrabutylammoniumazide in DMF. The azide groups are converted to amines by reaction withPtO₂ /H₂. The compound is then reacted with the N-hydroxysuccinimidederivative of 10, 12-pentacosadiynoic acid. Finally, the Boc group isremoved with trifluoroacetic acid to yield the 1N, 3N-bis (10,12-pentacosadiynoyl)-1, 2, 3-triaminopropane.

The lipids of the composition are crosslinked by activation appropriateto the type of polymerization chemistry employed. Diyne lipids arecross-linked by U.V. irradiation as originally described (Hub et al.,supra), monitoring visible absorption to follow the course of thereaction, which is usually complete by 20-60 min. Free radicalinitiators, where used, are removed from the preparation afterpolymerization by a suitable technique, such as dialysis.

Features of the Polymerized Lipid Compositions

One of the benefits of the crosslinked compositions is the ease by whichdifferent substituents can be screened and titrated for selectinbinding. The optimal proportion of a particular carbohydrate determinantand a particular anionic determinant are determined empirically bytitrating each substituent into the compositions and conducting asuitable selectin activity assay. This approach is illustrated furtherin Examples 2 and 3.

The proportion of lipids bearing a complex oligosaccharide like sLe^(x)is preferably between about 1% and 25%, preferably about 2% to 10%,optimally about 5%. Low values probably do not provide sufficientvalency, while higher values are believed to create steric problems forboth polymerization and binding accessibility. The proportion of lipidsbearing the electronegative determinant depends on the strength of thedeterminant. For many applications, there is no harm in using a hydroxylor carboxyl lipid for the balance of the lipid in the sheet. However,stronger acids may require more care. Excessive proportion of sulfate orphosphate may confer the composition with inhibitory activity for otherbiological reactions, particularly those that are naturally inhibited byhighly charged molecules, such as heparin. Where this is an issue, theproportion of such acids may be titrated down to a range of about 1% to50%, or 1% to 10%, or 0.5% to 2%, as appropriate.

The degree of polymerization between lipids in the lipid sheet is afactor of the proportion of lipids having crosslinkable substituents,and the completeness of the polymerization reaction. The practitionercan limit the amount of polymerization by including lipids in thepreparation that will not participate in crosslinking. While notintending to be bound by theory, it is a hypothesis of this disclosurethat the synergy between the carbohydrate and anionic components isimparted partly by the rigidity of the polymerized lipid structure. Itis recommended that at least 25%, preferably 50%, more preferably 75%,still more preferably 90%, even more preferably 95%, and still morepreferably almost 100% of the lipids in the sheet are crosslinked. Theentire sheet be polymerized into one unit, or into separate patches ortiles.

Where a proportion of non-reactive lipid is included as filler to reducethe degree of crosslinking, the carbohydrate determinant and the anionicdeterminant are typically on the crosslinked lipid rather than thefiller lipid. However, the opposite arrangement is possible, insofar asthe filler lipid will become entrapped by the neighboring crosslinks.Thus, in certain embodiments of the invention, either the carbohydratedeterminant or the anionic determinant for selectin binding, or both,are provided by non crosslinked lipids present in a lipid sheetcomprising other lipids that are crosslinked. This approach isespecially appropriate when using glycosphingolipids to satisfy thecarbohydrate determinant. Preferred lipids of this type aresulfoglucuronyl glycosphingolipids (Needham et al., Proc. Natl. Acad.Sci. USA 90:1359, 1993).

Both carbohydrate groups and electronegative groups are optionallyconjugated to the lipid through a spacer group. As will already beappreciated from the synthetic methods described earlier, hydrocarbonspacers of about 2 carbons in length provide a convenient approach toconjugation. In certain embodiments, the spacer groups are polyethyleneglycols that improve the stealth of the liposomes from uptake byreticuloendothelial cells. Since the anionic group and the carbohydrategroup must work in concert, the length of the spacer arms should match.The potency of polymerized lipid compositions is believed to derive inpart from the structural rigidity, and many embodiments have spacers ofminimal length.

In certain embodiments of this invention, a proportion of the lipids inthe lipid sheet have a first attached saccharide, and a separateproportion of the lipids have a second attached saccharide that isdifferent from the first. The two glycolipids are preferably part of thecross-linked structure. Embodiments where there is a higher plurality ofdifferent independently conjugated saccharides are contemplated. Anycombination of lipids in this arrangement that fulfills the carbohydratebinding requirement of selectins is suitable. In one example, the firstattached carbohydrate is an acidic monosaccharide such as sialic acid orsimilar sugar and the second carbohydrate is fucose or similar sugar.Combinations of lipids conjugated with different monosaccharides ordisaccharides or their analogs are of commercial interest because oftheir ease of synthesis.

Polymerized liposomes of this invention can be classified on the basisof their potency in various test assays known in the art. For example,when tested for inhibition of the binding of isolated selectin to cellsexpressing a selectin ligand such as PSGL-1, the liposomes preferablyare able to inhibit the binding in a manner that attains 50% maximalinhibition (IC₅₀) at a concentration of no more than about 10 TM,preferably no more than about 1 TM, still more preferably no more thanabout 100 nM, and even more preferably no more than about 10 nMoligosaccharide equivalents. A preferred binding assay of this type usesHL-60 cells, and is illustrated in Example 2. Polymerized liposomes mayalso be categorized in any assay on the basis of the relative IC₅₀compared with a suitable standard. The standard may be anoligosaccharide presented uncomplexed to liposomes or in a monomericform, such as sLe^(x) or sLe^(x) analog. The standard may also be aliposome having no oligosaccharide but otherwise the same lipidcomposition, or a liposome made with 100% carboxy terminated or hydroxyterminated lipids. In certain embodiments, the polymerized liposomeshave an IC₅₀ which is preferably 10² -fold lower, more preferably about10³ -fold lower, more preferably about 10⁴ -fold lower, still morepreferably about 10⁵ -fold lower, and even more preferably about 10⁶-fold lower than that of the standard.

This invention also includes embodiments which are selective for P- andL-selectin in comparison with E-selectin, or selective for P- orL-selectin in comparison with the other two selectins. A polymerizedliposome is selective if it has an IC₅₀ in an assay for inhibiting oneselectin that is higher than its IC₅₀ in an assay for inhibiting anotherselectin. An assay is preferably used for this determination that allowsthe particular selectin to be the only variable. The HL-60 selectinbinding assay outlined in Example 1 can be used for comparing P- andE-selectin inhibition using the same cells and switching chimeras. In asimilar fashion, the plated mucin in the ELISA described in Example 3binds a chimera of any of the three selectins, and can be used tocompare the inhibitory capacity of a particular composition for allthree selectins. Selective inhibitors preferably have an IC₅₀ that isabout 5-fold higher for the target selectin in comparison with anotherselectin; more preferably it is 25-fold higher; still more preferably itis 100-fold higher.

Inhibitors that are selective for P- and/or L-selectin are of particularinterest, because of recent observations that E-selectin antagonists canlead to conditions reminiscent of leukocyte adhesion deficiency disease(LAD-2), where neutrophils do not adhere normally to endothelialtissues, and recurrent bacterial infections of the lung, skin, andgingival tissues are common. Example 3 provides illustrations ofselective polymerized liposomes. Non-sulfated sugars like sLe^(x) andthe neutral disaccharides lactose and maltose are selective for L- andP-selectin when presented in the context of carboxy-terminated lipids.sLe^(x) is also selective in the context of hydroxyl-terminated lipids.Liposomes with sulfate groups either on sulfo Le^(x) or on a lipid incombination with sLe^(x) were not selective.

Also included are embodiments that are designed to optimize binding tomultiple selecting. These compositions may have a plurality of differentcarbohydrates and a plurality of different anionic or electronegativegroups on separate lipids.

Testing of the Polymerized Lipid Compositions

In vitro testing and optimization of the composition: Assays fordetermining the ability of a polymerized lipid composition to displayselectin ligands can be classified as either direct binding assays orinhibition assays.

Direct binding assays are conducted by permitting the composition tointeract directly with either a selectin or with a cell expressing aselectin. A lipid sheet containing various test selectin bindingdeterminants can be polymerized directly onto a microscope slide (Spevaket al., Adv. Mater. 7:85, 1995) and titrated with selectin, orconversely the selectin can be coated onto microtiter plate wells andtitrated with labeled polymerized lipid particles. Polymerized particlescan also be tested for direct binding to cells expressing selectinligands, such as HL-60 cells.

Since most of the applications for polymerized liposomes according tothis invention relate to an inhibition of binding between selectinligand-receptor pairs, it is more usual to develop and test compositionsin inhibition assays.

Inhibition capacity can be tested in cell-free assays where one memberof the selectin ligand-receptor pair is coupled to a solid surface, andthe second is presented for binding in the presence of the potentialinhibitor. After washing, the amount of second member bound isquantitated by way of a preattached or subsequently attached labelingsystem. This type of assay is convenient for comparative screening of anumber of different lipid compositions, for example, displayingdifferent carbohydrate and anionic determinants.

Many of the current cell-free selectin assay systems make use ofselectin chimeras, in which an N-terminal portion of the selectincomprising the binding domain is fused to a second protein fragment thatcan be used as an attachment means for a labeling system. A frequentlyused second fragment is an IgG Fc region, which can then be detectedusing a conjugate made with Protein A or anti-Fc. The construction ofchimeras and related assays are described by Watson et al. (J. CellBiol. 115:235, 1992), Aruffo et al. (Cell 67:35, 1991) and Foxall et al.(J. Cell Biol. 117:895, 1992).

One illustration of a convenient cell-free assay is the L-selectin ELISAdescribed in Bertozzi et al. (Biochemistry 34:14275, 1995). Briefly, acrude preparation of GlyCAM-1 is obtained from mouse serum. Microtiterplates are coated with polyclonal antibody specific for the peptidebackbone of the mucin, overlaid with the mucin, and then washed. Achimera of L-selectin fused to Fc is complexed with biotinylated F(ab')₂anti-Fc, which in turn is complexed to streptavidin-alkaline phosphataseconjugate. The combined conjugate is preincubated with the potentialinhibitor for 30 min, then transferred to the microtiter plate wells.After 30 min at room temperature, the wells are washed, and developedwith the enzyme substrate. In a variation of this type of assay,selectin ligand substitutes such as sulfatides are used that can becoated directly onto the plate. In another variation, the solidsubstrate is also a polymerized lipid (Spevak et al., Adv. Mater. 7:85,1995) expressing determinants that are at least as potent for selectinbinding as the compositions being tested as inhibitors.

Beyond the initial screening stage, one- or two-cell bioassays arepreferably used during the development of compositions as being morerepresentative of inhibition in a biological system.

A convenient one-cell assay for P-selectin inhibitors makes use of HL-60cells, available from the ATCC. HL-60 cells naturally express the PSGL-1antigen at about 36,000 sites per cell (Ushiyama et al., J. Biol. Chem.268:15229, 1993). The assay is described in Brandley et al. (Glycobiol.3:633, 1993). Briefly, an E or P-selectin chimera is incubated withbiotinylated goat F(ab'), anti-human IgG Fc, and an alkalinephosphatase-streptavidin conjugate for 30 min. This complex is thenincubated with potential inhibitors for ˜45 min at 37° C. 50 TL of themixture is added to each well of round-bottom microtiter platespreviously blocked with BSA. An equal volume of an HL-60 cell suspensionis added and the plate is incubated for 45 min at 4° C. Cells arepelleted to the well bottoms by centrifugation, washed, and developingusing p-nitrophenyl phosphate.

Other one-cell assays are done with cell isolates rather than celllines. The ability to inhibit neutrophil adhesion to purified P-selectinimmobilized on plastic wells can be determined using the assay describedby Geng, et al. (Nature 343:757, 1990). Briefly, human neutrophils areisolated from heparinized whole blood by density gradient centrifugationon Mono-Poly™ resolving media (Flow Laboratories), and suspended inHanks' balanced salt solution containing Ca⁺⁺, Mg⁺⁺, and human serumalbumin (HBSS/HSA). P-selectin is obtained by recombinant expression orisolated from outdated human platelet lysates by immunoaffinitychromatography on antibody S12-Sepharose™ and ion-exchangechromatography on a Mono-Q™ column (U.S. Pat. No. 5,464,935). TheP-selectin is coated onto microtiter plate wells at 5 Tg/mL. Cells areadded at ˜2×10⁵ per well, incubated at 22° C. for 20 min. The wells arethen filled with HBSS/HSA, sealed with acetate tape, and centrifuged.After discarding nonadherent cells and supernates, the contents of eachwell are solubilized with 0.5% hexadecyltrimethylammonium bromide inphosphate buffer and assayed for myeloperoxidase activity (Ley et al.,Blood 73:1324, 1989).

Two-cell adherence assays are conducted by testing the ability of acomposition to interfere with the attachment of one cell having aselectin to another cell having a ligand for the selectin. Oneillustration uses COS cells transfected to express the appropriateselectin (see generally Aruffo et al., Proc. Natl. Acad. Sci. USA84:8573, 1987). Transfected cell clones are selected for their abilityto support HL-60 cell adhesion. The clones are then expanded and grownin small-well culture plates as a substrate for the assay. Anothersuitable substrate cell are human umbilical vein endothelial cells(HUVEC), obtainable from Cell Systems, Inc., and stimulated with 100U/mL IL-1ν for 4 h. (Martens et al., J. Biol. Chem. 270:21129, 1995).HL-60 cells are labeled by incorporation of 1 TCi/mL [³ H]thymidine or10 Tg/mL calcein. The putative inhibitor is preincubated with thelabeled HL-60 cells, presented to the substrate cells, and then thewells are washed and counted.

Lymphocyte adherence can be determined using the frozen section assayoriginally described by Stamper et al. (J. Exp. Med. 144:, 828, 1976),since modified by Stoolman et al. (J. Cell Biol. 96:722, 1988), Arboneset al. (Immunity 1:247, 1994), and Brandley et al. (supra). Briefly,lymphocytes from mouse mesenteric lymph nodes or splenocytes arefluorescently labeled with CMFDA, and incubated with the test inhibitorfor ˜30 min at 0° C. The lymphocyte suspension is then overlaid on 10 Tmfrozen sections of mesenteric or peripheral lymph nodes (˜3×10⁴cells/section) and incubated on ice for 30 min on a rotator. Thesuspension is gently drained from the slide, and the sections are fixedwith 3% glutaraldehyde and counterstained with acridine orange. Fucoidancan be used as a positive control for inhibition. The adherence observedin this assay is attributable to L-selectin binding.

Leukocyte flow (rolling cell) assays are also described in Martens etal. (supra). Neutrophils are isolated from venous blood by dextransedimentation and Ficoll-Hypaque™ centrifugation. HUVEC are harvested bycollagenase treatment, plated onto 0.1% gelatin coated flasks, andcultured. A HUVEC monolayer is mounted on the flow chamber, and perfusedfor 2 min with buffer containing calcium and glucose. The isolatedneutrophils are preincubated with the test inhibitor in the same buffer.The neutrophil suspension is then passed over the HUVEC monolayer at awall shear stress of ˜1.85 dyne/cm². Interaction is videotaped for about10-20 min using a phase contrast microscope, and an imaging softwareprogram is used to determine the average number of neutrophils rollingon the monolayer in several different fields of view.

In vivo testing: Animal models for various diseases with an inflammatoryor immnunological etiology are known in the art and may be brought tobear in the testing of any composition that shows promising selectininhibitory action. In models of hyperacute disease such as reperfusioninjury, the composition is typically administered within minutes orhours of the inducing event to simulate a clinical setting. In models ofchronic disease, the composition is typically administered at regularperiods of a week or more during the progression phase. The animal isevaluated by cellular and clinical criteria for the ability of thecomposition to palliate the condition.

Amongst models suitable for the testing of selectin inhibitors are thefollowing: The cardiac ischemia reperfusion models of Weyrich et al. (J.Clin Invest. 91:2620, 1993), Murohara et al. (Cardiovasc. Res. 30:965,1995), Ma et al. (Circulation 88:649, 1993) Tojo et al. (Glycobiology6:463, 1996) and Garcia-Criado et al. (J. Am. Coll. Surg. 181:327,1995); the cardiact infarct model of Silver et al. (Circulation 92:492,1995); the pulmonary ischemia reperfusion models of Steinberg et al. (J.Heart Lung Transplant 13:306, 1994) and Kapelanski et al. (J. Heart LungTransplant 12:294, 1993); the cobra venom acute lung injury model andimmune complex lung inflammation model in U.S. Pat. No. 5,486,536; thehemmhoragic shock model of Kushimoto et al. (Thrombosis Res. 82:97,1996); the peritoneal exudate and endotoxin-induced uveitis models of WO96/35418; the bacterial peritonitis model of Sharar et al. (J. Immunol.151:4982, 1993); the meningitis model of Tang et al. (J. Clin. Invest.97:2485, 1996); the colitis model of Meenan et al. (Scand. J.Gastroenterol. 31:786, 1996); the Dacron graft experimental thrombusmodel of Palabrica et al. (Nature 359:848, 1992); the tumor metastasismodel of WO 96/34609; the allergic asthma model of WO 96/35418; theallergen mediated pulmonary hypersensitivity model of Gundel et al. (Am.Rev. Respir. Dis. 146:369, 1992); the diabetes models of Martin et al.(J. Autoimmunity 9:637, 1996) and Yang et al. (Proc. Natl. Acad. Sci.USA 90:10494, 1993); the model for immune complex alveolitis and dermalvasculitis by Mulligan et al. (J. Clin. Invest. 88:1393, 1991); thelymphocyte trafficking model of Hicke et al. (J. Clin. Invest. 98:2688,1996); the IgE-mediated skin reaction model of Wada et al. (J. Med.Chem. 39, 2055, 1996); and the collagen-induced arthritis anddelayed-type skin hypersensitivity models of Zeidler et al.(Autoimmunity 21:245, 1995). All the aforelisted descriptions of animalmodels are hereby incorporated herein by reference in their entirety.

Uses for Polymerized Liposomes

Research use: Polymerized lipid compositions of this invention can beused to characterize the nature of binding between putativeligand-receptor binding cells. For example, a newly isolated proteinreceptor that binds isolated neutrophils or HL-60 cells in a mannerinhibitible by liposomes this invention will be suspected as a selectin.A newly isolated mucin that binds HUVEC or cells transfected withselectin in a manner inhibitible by liposomes of this invention will besuspected of being a selectin ligand. Adhesion or activation of one cellby another in a manner inhibitible by liposomes of this invention willbe suspected of being mediated by selectin-ligand coupling.

Diagnostic use: Polymerized lipid compositions can also be used for thedetection of human disorders in which the ligands for the selectinsmight be defective. Such disorders would most likely be seen in patientswith increased susceptibility to infections involving an abnormality inleukocyte migration or lymphocyte activation.

For in vitro diagnostic procedures, cells to be tested are collectedfrom blood, separated by Ficoll-Hypaque™ centrifugation, and then testedfor their ability to bind a polymerized liposome with selectin bindingactivity. The liposome may be labeled with a radioisotopic orfluorescent marker, or if based on diyne chemistry, monitored by way ofits intrinsic color. Direct binding of the composition to the cells canprovide a measure of selectin on the cell surface. In one illustration,T cells or cells dispersed from a tumor biopsy are isolated and thecomposition is used to measure the density of selectin. In anotherillustration, the composition is used in a mixed leukocyte population tocount the number of cells expressing selectin.

For in vivo diagnostic procedures, the lipid composition is labeled byconjugation with or encapsulation of a suitable agent. Radioisotopessuch as ¹¹¹ In or ^(99m) Tc can be used as labels for scintigraphy, ornon-radioactive dense atoms can be used to enhance x-ray contrast. Thecomposition is administered intravenously at a peripheral site or vialocal intubation. Abnormal localization at a particular site may reflectunusual cell trafficking or activation with clinical implications.

Therapeutic use: Since the selectins have several functions related toleukocyte adherence, inflammation, and coagulation, compounds thatinterfere with binding of P-selectin or L-selectin can be used tomodulate the pathological consequences of these events.

An inflammatory response can cause damage to the host if unchecked,because leukocytes release many toxic molecules that can damage normaltissues. These molecules include proteolytic enzymes and free radicals.Examples of pathological situations in which leukocytes can cause tissuedamage include injury from ischemia and reperfusion, bacterial sepsisand disseminated intravascular coagulation, adult respiratory distresssyndrome, rheumatoid arthritis and atherosclerosis.

Reperfusion injury is a major problem in clinical cardiology.Therapeutic agents that reduce leukocyte adherence in ischemicmyocardium can significantly enhance the therapeutic efficacy ofthrombolytic agents. Thrombolytic therapy with agents such as tissueplasminogen activator or streptokinase can relieve coronary arteryobstruction in many patients with severe myocardial ischemia prior toirreversible myocardial cell death. However, many such patients stillsuffer myocardial necrosis despite restoration of blood flow.Reperfusion injury is known to be associated with adherence ofleukocytes to vascular endothelium in the ischemic zone, presumably inpart because of activation of platelets and endothelium by thrombin andcytokines that makes them adhesive for leukocytes (Romson et al.,Circulation 67:1016, 1983). The adherent leukocytes can migrate throughthe endothelium and destroy ischemic myocardium just as it is beingrescued by restoration of blood flow. Ischemia may occur pursuant to amyocardial infarction or as a result of complications of surgery, suchas deep vein thrombosis. Another inflammatory condition of concern incardiology is restenosis.

There are a number of other common clinical disorders in which ischemiaand reperfusion results in organ injury mediated by adherence ofleukocytes to vascular surfaces, including strokes; mesenteric andperipheral vascular disease; organ transplantation; and multiple organfailure following circulatory shock. Bacterial sepsis and disseminatedintravascular coagulation often exist concurrently in critically illpatients. These conditions are associated with generation of thrombin,cytokines, and other inflammatory mediators, activation of platelets andendothelium, and adherence of leukocytes and aggregation of plateletsthroughout the vascular system. Leukocyte-dependent organ damage is animportant feature of these conditions.

Adult respiratory distress syndrome is a devastating pulmonary disorderoccurring in patients with sepsis or following trauma, which isassociated with widespread adherence and aggregation of leukocytes inthe pulmonary circulation. This leads to extravasation of large amountsof plasma into the lungs and destruction of lung tissue, both mediatedin large part by leukocyte products.

Tumor cells from many malignancies (including carcinomas, lymphomas, andsarcomas) metastasize to distant sites through the vasculature. Themechanisms for adhesion of tumor cells to endothelium and theirsubsequent migration are not well understood, but may be similar tothose of leukocytes in at least some cases. The association of plateletswith metastasizing tumor cells has been well described, suggesting arole for platelets in the spread of some cancers. It has been reportedthat P-selectin binds to tumor cells in human carcinoma tissue sectionsand cell lines derived from carcinomas (Aruggo et al., Proc. Natl. Acad.Sci. USA 89:2292, 1992). In addition, certain tumors may themselvesexpress selectins or selectin ligands, which may participate in theadherence of metastasizing cells to endothelial cells or HEV at a newsite.

Antagonists of P-selectin may be beneficial for blockingplatelet-leukocyte interaction as thrombi develop (Welpy et al.,Biochim. Biophys. Acta 117:215, 1994). In baboons, administration ofanti P-selectin decreased fibrin deposition into Dacron graft implantswithout diminishing platelet accumulation into the grafts (Palabrica etat., Nature 359, 848, 1992). The results suggest that the trapping ofleukocytes, via interaction with platelets, may contribute to thedeposition of fibrin. Blocking P-selectin should prevent thisinteraction and nay have value as an anti-thrombogenic therapy.

To the extent that the initiation of an acute allograft or xenograftrejection involves selectin-mediated recruitment of inflammatory orimmune mediator cells, selectin antagonists can be brought to bear inthe few days after engraftment.

Antagonists of P- and L-selectin are also of interest in palliatingautoimmune diseases. For a review of the role of adhesion molecules inthese diseases, the reader is referred to Murray (Sernin. ArthritisRheum. 25:215, 1996).

Rheumatoid arthritis is characterized by symmetric, polyarticularinflammation of synovial-lined joints, and may involve extraarticulartissues, such as the pericardium, lung, and blood vessels. Adhesionmolecules appear to play an important role (Postigo et al., Autoimmunity16:69, 1993). Soluble selectins are present in the synovial fluid andblood of affected patients, correlating with elevated ESR and synovialPMN count (Carson CW et al. J. Rheumatol. 21:605, 1994). Conventionalantirheumatic therapy may modify synovial inflammation by alteringleukocyte adhesion. Corticosteroids, gold compounds, and colchicinedownregulate endothelial expression of selectins (Corkillet al, J.Rheumatol. 18:1453, 1991; Molad et al, Arthritis Rheum. 35:S35, 1992).

Systemic lupus erythematosus is characterized by formation ofantinuclear antibodies and manifest by inflammatory lesions on the skinand throughout the body. Selectin expression is increased on dermalvessel endothelial wall of patients with increased disease severity(Belmont et al., Arthritis Rheum. 37:376, 1994). Sjoren's syndrome,autoimmune thyroid disease, multiple sclerosis, and diabetes are otherconditions with a heavy implication of altered adhesion proteins such asICAM-1, LFA-1 and -3, VCAM-1, and selectins (Murray, supra), and may beamenable to therapy with selectin inhibitors.

Asthma is characterized by airway obstruction, inflammation, andincreased responsiveness to a variety of stimuli, manifest by episodesof cough, dyspnea and wheezing The steps proposed in chronic airwayinflammation include inflammatory stimulus triggering release ofmediators, followed by activation of the leukocyte-endothelial adhesioncascade resulting in leukocyte adhesion to the endothelium. Adhesionmolecules implicated include selectins, VCAM-1, and ICAM-1 which may beup-regulated following allergen challenge (Pilewski et al., Am. Rev.Respir. Dis 148, S31, 1993).

Timing and objectives of treatment. An effective amount of polymerizedlipid compositions may be used for treating an individual for conditionwherein etiology involves altered cell traffic or activation, mediatedin part by selectins.

An "individual" treated by the methods of this invention is avertebrate, particularly a mammal (including farm animals, sportanimals, and pets), and typically a human.

"Treatment" refers to clinical intervention in an attempt to alter thenatural course of the individual being treated, and may be performedeither for prophylaxis or during the course of clinical pathology.Desirable effects include preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, such as hyperresponsiveness,inflammation, or necrosis, preventing metastasis, lowering the rate ofdisease progression, amelioration or palliation of the disease state,and remission or improved prognosis. The "pathology" associated with adisease condition is anything that compromises the well-being, normalphysiology, or quality of life of the affected individual.

Treatment is performed by administering an effective amount of apolymerized lipid composition of this invention. An "effective amount"is an amount sufficient to effect a beneficial or desired clinicalresult, and can be administered in one or more doses.

The modes of treatment contemplated in this invention include but arenot limited to the following:

1. Inhibiting leukocyte adhesion or migration, comprising administeringa P-selectin inhibitor so as to inhibit binding between a vascularendothelial cell and a leukocyte selected from the group consisting ofneutrophils, monocytes, eosinophits, and lymphocytes bearing aP-selectin ligand, thought to be memory T cells. The inhibiting can beperformed either by introducing the inhibitor into an environment wherethe interacting cells come into contact, particularly near the affectedsite, or contacting the cell bearing the selectin with the inhibitor inthe absence of the cell bearing the ligand.

2. Inhibiting platelet aggregation or fibrin deposition by administeringa P-selectin inhibitor to an environment containing platelets orsusceptible of accumulating platelets.

3. Inhibiting leukocyte adhesion or migration, comprising administeringan L-selectin inhibitor so as to inhibit binding between a lymphocyte,neutrophil or monocyte and an endothelial cell or lymphatic tissue,particularly an HEV cell.

4. Inhibiting lymphocyte adhesion, migration, or activation, comprisingadministering an L-selectin inhibitor to the lymphocyte.

5. Inhibiting metastasis of a tumor suspected of expressing a selectinligand or receptor by administering an inhibitor for the selectin to thetumor or to the circulation.

The criteria for assessing response to therapeutic modalities employingthe lipid compositions of this invention are dictated by the specificcondition, For example, the treatment to prevent extension of myocardialinfarction can be monitored by serial determination of marker enzymesfor myocardial necrosis, and by EKG, vital signs, and clinical response.Treatment of acute respiratory distress syndrome can be monitored byfollowing arterial oxygen levels, resolution of pulmonary infiltrates,and clinical improvement as measured by lessened dyspnea and tachypnea.Other conditions treated using the methods of this invention aremeasured according to standard medical procedures appropriate for thecondition.

Pharmaceutical preparations and administration: Compositions preparedfor use according to this invention can be prepared for administrationto an individual in need thereof, particularly humans, in accordancewith generally accepted procedures for the preparation of pharmaceuticalcompositions. Preferred methods for preparing liposomes described hereinare sufficiently flexible that batch sizes from 5 mL to several litersor more can be prepared reproducibly and under sterile conditions.

General procedures for preparing pharmaceutical compositions aredescribed in Remington's Pharmaceutical Sciences, E. W. Martin ed, MackPublishing Co., Pennsylvania. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dispersing a liposome in aliquid excipient, such as water, saline, aqueous dextrose, or glycerol.The liposome suspension may include lipid-protective agents to protectlipids against free-radical and lipid-peroxidative damages on storage.Lipophilic free-radical quenchers, such as alphatocopherol andwater-soluble iron-specific chelators, such as ferrioxarnine, can beused. One of the advantages of the polymerized lipid compositions of thepresent invention is stability against many of the usual degradativeeffects that accumulate upon storage. The composition may optionallyalso contain other medicinal agents, pharmaceutical agents, andcarriers.

Compositions for injection can be supplied as liquid solutions orsuspensions, or as solid forms suitable for dissolution or suspension inliquid prior to injection. For administration to the trachea andbronchial epithelium, a preferred composition is one that provideseither a solid or liquid aerosol when used with an appropriateaerosolizer device. Although not required, pharmaceutical compositionsare in some instances supplied in unit dosage form suitable foradministration of a precise amount.

The route of administration of a pharmaceutical composition depends,inter alia, on the intended target site, clinical condition, and thenature of the condition being treated. Intravenous or intraly mphoidadministration or injection directly into an affected site are the mostusual routes. Pulmonary administration by aerosol is conducted using anebulizer device. Apparatus and methods for forming aerosols aredescribed in Kirk-Othmer, "Encyclopedia of Chemical Technology", 4the EdVol. 1, Wiley N.Y., pp. 670-685, 1991.

The size of the dose is selected taking into account the expected volumeof distribution of the composition before reaching the intended site ofaction, and then providing sufficient inhibitor (in nM sugar equivalent)to meet or exceed the IC₅₀ concentration as measured in an appropriatecell bioassay, typically at about 2-20 times IC₅₀ concentration. Inplanning the dose, it may not be necessary to completely block all ofthe selectin receptors. For normal healing, at least some leukocytes mayneed to migrate to the affected site. The amount of inhibitor isadjusted accordingly.

The assessment of the clinical features and the design of an appropriatetherapeutic regimen for the individual patient is ultimately theresponsibility of the prescribing physician.

The foregoing description provides, inter alia, a detailed explanationof how polymerized lipid compositions can be used to inhibit cellularevents mediated by selectin binding. It is understood that variationsmay be made with respect to structure of the composition or itsimplementation without departing from the spirit of this invention.

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference in their entirety.

The examples presented below are provided as a further guide to apractitioner of ordinary skill in the art, and are not meant to belimiting in any way.

EXAMPLES Example 1

Development of Two-Component Glycoliposomes

Glycoliposomes were formed by attaching a carbohydrate component to apolymerizable lipid, mixing with a second polymerizable lipid with apolar head group, forming liposomes, and then polymerizing the lipids.

FIG. 3 shows the sialyl Lewis^(x) (sLe^(x)) tetrasaccharide(structure 1) in comparison with the components assembled intoliposomes. The carbohydrates labeled as 2a (an sLe^(x) analog), 3a(lactose), and 4a (maltose) were used for synthesizing the polymerizableglycolipids, hereafter designated as 2b, 3b and 4b, respectively. Theprecursor polymerizable lipid was 10,12-pentacosadiynoic acid (PDA),which was conjugated to the carbohydrate by standard techniques. Thesecond polymerizable lipid used during liposome formation was eithercompound 5 (PDA), which comprises a negatively charged headgroup, orcompound 6, which comprises a polar but uncharged headgroup.

FIG. 1 depicts an expanded view of polymerized glycoliposomes,containing either compounds 2b and 5(A) or 2b and 6(B). The polymerizedglycoliposomes were formed as follows: Various molar percentages oflipids were prepared so as to give 1 mM solutions in total lipid whilevarying the percentages of glycolipids in the range 0.5 to 50%. Theglycolipids were formed into liposomes by the probe sonication method(R.R.C. New, pp. 33-104, in "Liposomes: a practical approach", Oxford U.Press 1990). The lipids appeared to be miscible based on an analysis oftheir Langmuir isotherms (G. L. Gaines, in "Insoluble monolayers atliquid-gas interfaces", Wiley:N.Y. 1966).

Polymerization of the liposomes was carried out by exposure of theaqueous solutions to UV light at 254 nm (Hub et al., Angew. Chem. Int.Ed. Engl. 19:938, 1980; Spevak et al., J. Amer. Chem. Soc. 115:1146,1993). Polymerization of lipid diacetylenes requires the monomers toadopt a solid analogous state. The carbohydrate percentages reportedhere are estimates of the sugar groups appearing on both the inner andouter liposome surfaces. With percentages of the glycolipid componentabove approximately 40%, polymerization was substantially inhibited.This is rationalized by the steric crowding of adjacent carbohydrateheadgroups which prevent the proximal diacetylenes from polymerizing.

Characterization of the polymerized glycoliposomes by transmissionelectron microscopy (TEM) showed that the preparation consisted ofspheres between 20-100 nm in diameter.

Example 2

Bioassay for Selectin Inhibition Activity

Ability of the compositions prepared in Example 1 to inhibit selectinbinding was tested in a standard bioassay. The assay for measuringP-selectin binding to HL-60 cells was taken from the description inBrandley et al. (Glycobiol. 3:633, 1993). Briefly, P-selectin chimera isallowed to form a complex with biotinylated goat F(ab') anti-human IgGFc and alkaline phosphatase-streptavidin, and is preincubated withinhibitors before mixing with HL-60 cells. The cells were pelleted bycentrifugation and washed with TBS. Chromagen was added and the colorthat developed was read as an OD at 405 nm. All assays were run inquadruplicate.

FIG. 4 shows the inhibition titration curve for various polymerizedglycoliposome preparations containing 5% carbohydrate-linked lipid. Opentriangles: sLe^(x) analog conjugate plus acidic lipids. Open circles:sLe^(x) analog conjugate plus neutral lipids. Closed circles: lactoseconjugate plus acidic lipids. Squares: maltose conjugate plus acidiclipids. It is evident from the results of this assay that the presenceof the acidic lipid is critical for measurable inhibition, even when themost effective carbohydrate conjugate of those tested, the sLe^(x)analog, is used. The neutral disaccharides lactose and maltose also haveselectin inhibition activity when used alongside acidic lipids. All thecompositions having a saccharide and a negatively charged lipidinhibited P-selectin binding in a dose-dependent fashion.

FIGS. 5A and 5B show the concentration giving 50% inhibition (IC₅₀) forvarious polymerized glycolipid compositions. The IC₅₀ values are basedon the total concentration of glycolipid. No reduction was made for anyglycoside that may be inaccessible due to incorporation into the innerlayer of the liposome. Therefore, these IC₅₀ values represent an upperlimit of the actual glycoside available for binding.

FIG. 5A is a titration analysis of the optimal proportion ofcarbohydrate lipid to total lipid in the composition. This experimentwas conducted with the sLe^(x) analog lipid conjugate, with the balanceof the composition being the lipid having the carboxylic acid headgroup.It is evident that the optimal percentage is about 5%, althoughcompositions up to at least 50% contain inhibitory activity, andcompositions up to about 20% have inhibitory activity in the nM range.The decrease in inhibitory activity at the higher percentages correlateswith the increased difficulty in polymerizing these compositions, whichis attributed to steric hindrance by the carbohydrate. The 2 nM IC₅₀ forthe 5% composition contrasts by about 1 to 5×10⁶ with values obtained inthis assay for sLe^(x) monomer.

FIG. 5B is a comparison of the IC₅₀ for various compositions withdifferent carbohydrate constituents. Both lactose and maltose providesignificant inhibitory activity (15 nM and 200 nM respectively) whenprovided in the context of acidic lipids. The value for lactose inparticular compares favorably with that for sLe^(x) compositions. Thelast two bars show the lack of detectable inhibition by polymerizedliposomes made with acidic or neutral lipids alone.

Thus, both a suitable carbohydrate and a separate negatively chargedlipid are required in these preparations to provide selectin inhibitionactivity. In hindsight, we speculate that the binding of otherinhibitory compounds, such as certain types of heparin, inositol hexakisphosphate, sulphoglucuronyl glycolipids, fucoidan, sulfatides and ansLe^(x) -RGD conjugate, can be explained as a combination of acarbohydrate or carbohydrate-like molecules and separately spacedmultiple acid groups.

The possibility of intercalation of the liposomes into the cells,thereby effecting their ability to bind P-selectin, was also addressed.The cells were pretreated with the liposomes and washed to remove theliposomes prior to the addition of the P-selectin chimera. This did notresult in any reduction in selectin binding to the cells. The inhibitionwas unaffected in experiments where the reagents and inhibitors wereadded simultaneously to the microtiter plates.

By way of comparison, the level of sLe^(x) or sLe^(x) analog presentedas a monomer required to reach IC₅₀ in this assay is ˜1 to 5 mm. Therelative improvement imparted by incorporation in the polymerizedliposome is approximately 10⁶ -fold.

Example 3

Further Confirmation of the Requirement for Negatively Charged Lipids

Additional polymerized glycoliposome compositions were prepared fortesting in a different assay system.

The assay is an ELISA in which the polymerized liposomes are tested foran ability to inhibit the binding of selectin chimera to isolatedGlyCAM-1. A full description is provided in Bertozzi et al.(Biochemistry 34:14275, 1995). Briefly, a crude preparation of GlyCAM-1is obtained from mouse serum by extraction with 2:1 chloroform/methanol,recovery of the aqueous phase, and concentration. This mucin acts inthis assay as a ligand for any of the three selecting. Microtiter platesare coated with polyclonal antibody specific for the peptide backbone ofthe mucin, overlaid with the mucin, and then washed. Meanwhile, acomplex is formed between: a) a chimera of the respective selectin fusedto the Fc region of the human IgG heavy chain; b) biotinylated F(ab')₂anti-Fc; c) streptavidin-alkaline phosphatase conjugate. This solution(70 TL) is combined with 70 TL of inhibitor and incubated for 30 min,then transferred to the microtiter plate wells. After 30 min at roomtemperature, the wells are washed, and developed with the enzymesubstrate p-nitrophenyl phosphate.

FIGS. 6A and 6B show the polymerized liposomes prepared for testing.Five different groups were prepared having either no oligosaccharide(Group 1, depicted in FIG. 6A), or one of four different oligosaccharideconjugated lipids at a relative molar concentration of 5% (Groups 2-5,depicted in FIGS. 6B-6E respectively). Within each group, thesubstituent on the lipids not conjugated with oligosaccharide (shown asan "X" in the diagram) was varied as follows:

an amine, which has a positive charge at neutral pH;

a hydroxyl group, which is neutral but electronegative;

a carboxylic acid, which has a negative charge at neutral pH; or

a mixture comprising either 5% or 50% lipid with the oxyacid sulfate,the balance being lipid with a hydroxyl head group.

These compositions gave the following results in the selectin inhibitionassay:

                                      TABLE 1                                     __________________________________________________________________________    Selectin Inhibition of Polymerized Glycoliposomes                                                       Inhibitory Activity                                   IC.sub.50 in TM                                                                 Carbohydrate lipid    L-  E-   P-                                           Group substituent Other lipid substituent selectin selectin selectin        __________________________________________________________________________    1   (none)   --CONHCH.sub.2 CH.sub.2 NH.sub.2                                                           >250                                                                              >250 >250                                           --CONHCH.sub.2 CH.sub.2 OH >250 >250 >50                                      --COOH >250 >250 >100                                                         --CONHCH.sub.2 CH.sub.2 OSO.sub.3 -- >250 >250 18                             --CONHCH.sub.2 CH.sub.2 OH(5:95)                                              --CONHCH.sub.2 CH.sub.2 OSO.sub.3 -- 7.5 >50 4.4                              --CONHCH.sub.2 CH.sub.2 OH (50:50)                                          2 5% sLe.sup.x analog --CONHCH.sub.2 CH.sub.2 NH.sub.2 >12.5 >12.5                                             >12.5                                          --CONHCH.sub.2 CH.sub.2 OH 1.12 >12.5 1.5                                     --COOH 0.50 >2.5 0.47                                                         --CONHCH.sub.2 CH.sub.2 OSO.sub.3 -- 0.26 0.45 0.18                           --CONHCH.sub.2 CH.sub.2 OH (50:50)                                          3 5% sulfo Le.sup.x --CONHCH.sub.2 CH.sub.2 NH.sub.2 >12.5 >12.5 >12.5                                           analog --CONHCH.sub.2 CH.sub.2 OH                                           0.26 0.38 0.18                                 --COOH 0.26 0.68 0.28                                                         --CONHCH.sub.2 CH.sub.2 OSO.sub.3 -- 0.20 n.d. n.d.                           --CONHCH.sub.2 CH.sub.2 OH (50:50)                                          4 5% lactose --CONHCH.sub.2 CH.sub.2 OH >12.5 >12.5 >12.5                       --COOH 1.80 >12.5 0.50                                                      5 5% maltose --CONHCH.sub.2 CH.sub.2 OH >12.5 >12.5 3.0                         --COOH 3.0 >12.5 1.3                                                      __________________________________________________________________________

The IC₅₀ values are all based on the total amount of liposome boundcarbohydrate except in Group 1, where the values are calculated from thetotal amount of matrix head groups.

The results support the following conclusions. First, the sulfatedcarbohydrate sulfo Le^(x) analog has a very low IC₅₀ (high inhibitorycapacity) for L-, E- or P-selectin in a context of acidic or polarlipids (but not positively charged lipids). Where the saccharide is thenon-sulfated sLe^(x) analog, an acidic neighboring lipid is required forfull inhibitory activity, which is selective for L- and P-selectin.Sulfate lipids support sLe^(x) binding better than carboxylate lipids,even at a relative proportion of 50%. As in the preceding example, thepresence of acid lipids turn ineffective neutral disaccharides likelactose and maltose into effective inhibitors. This effect occurred onlyfor L- and P-selectin, since none of the neutral disaccharidecompositions inhibited E-selectin binding. The contributory effect ofacid groups to the binding of L- and P-selectin is consistent with theworking hypothesis that the lipid acid groups fulfill a selectin bindingrequirement equivalent to what is provided by sulfotyrosine or itsequivalent in the biological ligands.

This mixed construction approach combined with a simple plate-bindingassay provides a rapid method for identifying carbohydrate-acid groupcombinations that are capable of selectively inhibiting the binding ofdifferent selectin-ligand pairs.

Example 4

Cell activity Assays Confirm Biological Efficacy of Glycoliposomes

Glycoliposomes containing 5% sulfo Le^(x) analog and 95%hydroxyl-terminated lipid were tested in a flow adhesion assay (Alon etal., Nature 374:539, 1995). Briefly, P-selectin chimera is immobilizedin a flow chamber and the affinity of HL-60 cells for this substrate ismanifest for their ability to roll slowly along on the surface. Theinteraction is specific for the PSGL-1 mucin domain on the HL-60 cellsand the inhibitor's ability to block cell adhesion under physiologicalflow rather than under static conditions. At a glycolipid concentrationof 1 TM, this glycoliposome formulation was able to completely inhibitHL-60 cell rolling on P-selectin surfaces. The control liposome (withoutthe carbohydrate) had no effect.

The same liposome formulation was tested in the Stamper-Woodrufflymphocyte homing assay (Stamper et al., J. Exp. Med. 144:828, 1976).This assay measures ability of lymphocytes to home into lymph nodesthrough high endothelial venules (HEV), a process known to be mediatedby L-selectin. Thoracic duct lymphocytes (TDC) were counted on fixedsections of HEV in the presence of the liposomes. The 5% glycoliposomecompletely inhibited the TDC from binding to HEV at a concentration of 1TM. The control liposome had no effect.

Example 5

Alternative Saccharide Components

Further refinement of the carbohydrate component of polymerizedliposomes is conducted along several fronts.

In one experimental series, the prototype oligosaccharides sLe^(x) andsulfo Le^(x) are dissected into various substituents and tested inindependent compositions. FIG. 7 shows some monosaccharide anddisaccharide lipid conjugates of interest. Other saccharides of interestare lactosamine, 3' sialyl lactosamine, and 3' sialyl lactose. Theidentification of active subcomponent saccharides has two purposes. Oneis to further elucidate the binding requirements for each selectin,which can then be used to develop inexpensive analog structures withenhanced binding or selectin specificity. Another is to identify mono-and disaccharides that can be used in a mixed saccharide liposome, asexplained below.

In another experimental series, other oligosaccharides believed to haveequal or better activity than the prototypes as monomers are tested inliposome compositions to determine if the activity can be enhancedfurther. Conjugates of interest are shown in FIG. 8. Other conjugates ofinterest are various sLe^(x) analongs and other structures listedelsewhere in the disclosure.

The conjugates are formed similarly to those in the previous examples:by formation of peracetylated beta-NAc-allyl glycoside, combined withcystamine hydrochloride under U.V. light, and then coupled directly withthe activated acid of PDA.

Mixed saccharide liposomes have different saccharides conjugated todifferent lipids in the composition. It is proposed that the differentsaccharides can work in concert to supply the carbohydrate requirementfor selectin binding, when presented in the context of other lipidssatisfying the anionic binding requirement in a polymerized lipid sheet.Of particular interest is a combination of sialic acid and fucose, sincethese are believed to be the residues in sLe^(x) responsible forselectin binding.

The sialic acid conjugate is prepared according to the standard methodoutlined in Spevak et al. J. Amer. Chem. Soc. (1993) 115, 1146. Thefucose conjugate is prepared as follows. First, the perbenzoylated,glycosylchloride of fucose is C-allylated by trimethylallylsilane andtrimethylsilyltriflate (Hosomi et al. Tetrahedron Lett. 2383, 1984 togive the C-glycoside. This compound is deprotected by sodium/ammonia.The perbenzoylated C-glycoside of fucose is dissolved in t-butanol andadded to refluxing, anhydrous ammonia, protected from moisture. Solidsodium metal is then added until the blue color persists for at least 20min. The reaction is then quenched with ammonium chloride and theammonia is allowed to evaporate. The solid residue is dissolved inwater, brought to about pH 2 with conc. HCl and extracted with ethylacetate several times. The combined organic solutions are dried withmagnesium sulfate and filtered. After evaporation the C-glycosideproduct is purified by flash chromatography. The C-glycoside isdissolved with cystamine-hydrochloride (3 eq.) in degassed water to givea 1 molar sugar solution. The solution is kept under a constant blanketof nitrogen and irradiated with UV light (254 nm). After 12 hours thesolution is neutralized with solid sodium bicarbonate, concentrated andflash chromatographed yielding the amine. This is dissolved in a minimalamount of methanol, added to this solution of NHS-PDA (1.2 eq.) andstirred for 12 hrs. The reaction is diluted with chloroform, washed withsaturated, aqueous sodium bicarbonate, then dried with magnesium sulfateand filtered. After evaporation, the crude glycolipid residue ispurified by flash chromatography.

Sialic acid conjugate and fucose conjugate are mixed at a ratio of 1:1and then combined with PDA at 5 to 10% glycoconjugate as molar percentof total lipid. Vesicle formation and lipid polymerization proceed asnormal to form a mixed glycoliposome with a surface structure shown inFIG. 9.

The polymerized lipid compositions described in this example are testedaccording to the assay described in Example 3.

Example 6

Therapeutic Testing in Animal Models

Polymerized liposome compositions having good inhibition activity inselectin binding assays are tested further for their efficacy in diseasemodels. All trials are conduced in accordance and with the approval ofthe appropriate Animal Use Committee.

Myocardial ischemia and reperfusion injury are modeled according toprotocols similar to those of Weyrich et al. (J. Clin. Invest. 91:2620,1993), Murohara et al. (Cardiovascular Res. 30:965, 1995), and Ma et al.(Circulation 88:649, 1993). Briefly, adult mammals of a higher species(typically canine, feline, or ovine) are anesthetized, and a midstemalthoracotomy is performed. A silk ligature is tied around the leftanterior descending coronary artery 8-10 mm from the origin. EKG andMABP are continuously monitored. The animals are allowed to stabilize,and then myocardial ischemia (MI) is induced by tightening the ligatureto complete occlusion. The test therapeutic agent is given intravenouslyas a bolus 80 min later. At the 90 min time point, the ligature isuntied and the myocardium is allowed to reperfuse for 270 min.

The ligature is retightened, and the aria at risk is identified byinjecting Evans blue. After excision, irreversibly injured parts of theheart are identified by dissection and staining using 0.1% nitrobluetetrazolium, and calculated as a percentage of mass of the organ. Theproportional area effected is reduced upon successful treatment.Myeloperoxidase activity in the ischemic myocardium as determinedaccording to Mullane et al. (J. Pharmacol. Meth. 14:157, 1985) ispreferably also reduced. The ischemic-reperfused coronary endotheliumcan also be measured for adherence of isolated autologous PMN, and ispreferably reduced in the treated animals. The animals are tested inthree groups: MI induced and inhibitor treated; MI induced and controltreated; and sham MI (operated but without vessel occlusion).

Treated animals in the cited studies responded to 400 Tg/kg of sLe^(x)presented as a phospholipid liposome, or 1 mg/kg of the anti-L-selectinmonoclonal antibody DREG-200. In the present experiment, polymerizedliposomes are tested in a range of about 10-400 Tg of carbohydrateequivalent per kg body weight. An equal number of polymerized liposomesmade of 100% neutral lipids is given at an equal dose (on a per-liposomebasis) as vehicle control. To the extent that necrosis induced by othertypes of acute cardiac inflammatory events, such as myocarditis,restenosis and deep vein thrombosis, are mediated by similar mechanisms,the effective doses established in the cardiac reperfusion model mayalso be considered for these conditions.

Lung reperfusion injury is modeled according to protocols similar tothose of Steinberg et al. (J. Heart Lung Transplant 13:306, 1994) andKapelanski et al. (J. Heart Lung Transplant 12:294, 1993). Briefly,general anesthesia is induced, and the left lung is exposed by excisionof adjacent ribs, intercostal muscles and neurovasculature. After a 30min recovery period, animals are selected for continuation that have anarterial oxygen tension above 200 mm Hg and a carbondioxide tensionbelow 45 mm Hg. After systemic heparinization, ischemia of the left lungis initiated by occlusion of the left main pulmonary artery. The periodof ischemia is about 3 h, whereupon the lung is ventilated and permittedto reperfuse. Ten minutes before reperfusion, animals receive a bolusintravenous infusion of the test therapeutic compound. Ten minutes afterthe onset of reperfusion, the right pulmonary artery is ligated, and thetip of an endotracheal tube is advanced beyond the orifice of thetrachial bronchus, and the right main bronchus is clamped at endexpiration. Physiologic parameters are recorded for 6 h. Animals arecompared on the basis of survival data, plus several of the following:gravimetric lung water, partial pressures of oxygen and carbon dioxide,inert gas shunt, pulmonary vascular resistance, and circulating WBC,neutrophil and lymphocyte count. Sham animals are operated but thepulmonary artery is not ligated--both lungs are ventilated during the 3h period, and then worked up as in the test animals.

In the first study cited above, ischemic animals responded to 1 mg/kg ofthe monoclonal antibody EL-246, specific for L- and E-selectin. In thepresent experiment, polymerized liposomes are tested in a range of about10-400 Tg of carbohydrate equivalent per kg body weight. An equal numberof polymerized liposomes made of 100% neutral lipids is given at anequal dose (on a per-liposome basis) as vehicle control.

Pulmonary vascular injury induced by hemorrhagic shock is modeledaccording to protocols similar to those of Kushimoto et al. (ThrombosisRes. 82:97, 1996). Briefly, adult rats are anesthetized withpentobarbital, the right carotid artery is cannulated for monitoringblood pressure, and the left femoral artery is cannulated for samplingblood and administering fluids. Phlebotomy is induced by gradualwithdrawal of 25 mL blood/kg over 15 min using a syringe pump. The meanarterial pressure is maintained between ˜30-40 mm Hg for 30 min, andthen the rats are resuscitated with 75 mL/kg lactated Ringer's solution,infused over 30 min. Physiological body temperature was maintainedduring this procedure using a heat lamp. Sham animals are cannulated inthe same fashion, but no blood is removed. Pulmonary accumulation ofleukocytes, measured as myeloperoxidase activity, and pulmonary vascularpermeability to bovine serum albumin (BSA) peaks at 6 h. The hemorrhagicshock is reversible, because animals surviving the first 6 h and allowedto recover survive for at least another 5 days.

The therapeutic compound is tested by administering boluses of testcompound through the femoral artery cannula at regular intervals throughthe critical period (0, 2, and 4 h following fluid resuscitation). ¹²⁵I-BSA is injected 30 min prior to sacrifice at the 6 h point. A midlinelaparotomy is performed, blood is withdrawn from the abdominal aorta,and the pulmonary vasculature is perfused with saline via rightventricular puncture. Pulmonary vascular permeability is calculated as aratio cpm in lung versus plasma, and is an indication of pulmonaryvascular damage. Lung samples are homogenized and assayed formyeloperoxidase activity according to Warren et al. (J. Clin. Invest.84:1873, 1989), as an indication of the number of neutrophils in thelung. Reduction of myeloperoxidase activity and/or permeability by thetest composition compared with vehicle control is an indication ofefficacy.

In the cited study, hemorrhagic animals responded to 1 mg/kg of themonoclonal antibody PB 1.3. In the present experiment, polymerizedliposomes are tested in a range of about 10-400 Tg of carbohydrateequivalent per kg body weight per administration.

Tumor metastasis is modeled according to protocols similar to thosedescribed in PCT application WO 96/34609. This model is based on thehighly metastatic BL6 clone of the B16 melanoma cell line (Dr. JeanStarkey, Montana Stane U., Bozeman Mont.), or a similar line establishedand cloned by standard techniques from an excised melanoma or carcinoma.A suspension of metastatic cells is suspended and incubated for 5-10 minat 37° C. with the therapeutic test compound at various concentrations,or a vehicle control. Following incubation, about 2-5×10⁴ cells in avolume of 200 TL are injected into the tail vein of 8 week old syngeneicmice. After about 3 weeks, the animals are sacrificed. Lung and liverare excised and fixed in 10% formaldehyde, and tumor cell colonies arecounted under a dissecting microscope. Colonies with a diameter >1 mmare counted separately from smaller colonies. A positive result isindicated by a substantial reduction in the total number of colonies orin the proportion of larger colonies. Polymerized liposome preparationsare tested in a range of 5 nM-10 TM final concentration of carbohydrateequivalent in the cell incubation mixture.

Allergic asthma is modeled according to protocols similar to thosedescribed in PCT application WO 96/35418. Briefly, adult sheep areselected on the basis of having an established early and late bronchialresponse to inhaled Ascaris suum antigen. Animals are restrained, andthe nasal passages are topically anesthetized with lidocane. The animalsare intubated with a cuffed endotracheal tube through the oppositenostril with a flexible fiber optic bronchoscope as guide. Pleuralpressure is estimated with an esophageal balloon catheter. Lateralpressure is measured with a sidehole catheter (i.d. 2.5 mm) advancedthrough and positioned distal to the tip of the endotracheal tube. Thetracheal and pleural pressure catheters are connected to a differentialpressure transducer for measuring transpulmonary pressure. Airflow ismeasured by connecting the proximal end of the endotracheal tube to apneumotachograph. Pulmonary flow resistance is calculated as the changein transpulmonary pressure divided by the change in flow at mid-tidalvolume, averaged over 5 breaths. Thoracic gas volume is measured in aconstant-volume body plethysmograph to obtain specific lung resistance(SR_(L)).

Aerosols of test therapeutic suspensions are generated using a nebulizerthat provides a median aerodynamic diameter of ˜3 Tm. The nebulizer isconnected to a dosimeter system, consisting of a solenoid valve and asource of compressed air. The solenoid valve is activated for 1 sec atthe beginning of the inspiratory cycle of the respirator. Aerosols aredelivered at a tidal volume of 500 mL at a rate of 20 breaths perminute. The test therapeutic compound is administered via nebulizer. Toassess bronchial responsiveness, cumulative concentration responsecurves are determined by measuring SR_(L) immediately after inhalationof buffer, and after each consecutive administration of 10 breaths ofincreasing concentrations of carbachol, in the range of ˜0.25% to ˜4%(wt/vol). The test is discontinued when SR_(L) exceeds 400% of initialvalue or the maximal dose is reached. Bronchial responsiveness isassessed by determining the point at which SR_(L) reached 400%.Polymerized liposome preparations are tested in a range of 5 nM-10 TMfinal concentration of carbohydrate equivalent in the aerosol solution.

Arthritis is modeled according to the collagen type-II induced arthritismodel of Zeidler et al. (Autoimmunity 21:245, 1995). Briefly, groups ofage-matched DBA/1 mice are immunized intradermally with 100 Tg collagentype II from bovine cartilage, emulsified in complete Freund's adjuvant,followed 18 days later with 50 Tg in incomplete Freund's adjuvant. Testtherapeutic compositions are administered weekly from about week 4 toabout week 8 following the first collagen injection. The disease isassessed daily by visual signs of erythema, and of swelling of one ormore joints. Immunological signs of autoimmunity are monitored bystandard immunoassays for serum antibody against collagen type II,collagen type I, and proteoglycans. Reduction in the titers of theautoantibodies, or a delay in the appearance of visual signs ofarthritis, are indications of efficacy. Polymerized liposomes are testedin a range of about 10-400 Tg of carbohydrate equivalent per kg bodyweight. In the present experiment, polymerized liposomes are tested in arange of about 10-400 Tg of carbohydrate equivalent per kg body weightper administration, or an equal number of control liposomes.

Other established animal models are implemented in the testing ofpolymerized liposomes for the treatment of additional clinicalconditions of interest.

What is claimed as the invention is:
 1. A composition for inhibitingbinding between a first cell having a P- or L-selectin and a second cellhaving a ligand for the selectin, comprising a sheet of lipids wherein aproportion of the lipids sufficient to stabilize the sheet arecovalently crosslinked, a proportion of the lipids have an attachedsaccharide which meets the carbohydrate binding requirements ofselectins, and a proportion of the lipids not having an attachedsaccharide have an acid group that is negetively charged at neutral pHwhich meets the anionic binding requirement of P- and L-selectin.
 2. Thecomposition of claim 1, wherein the saccharide is a sialylatedfucooligosaccharide or analog thereof.
 3. The composition of claim 1,wherein the saccharide is a sulfated fucoohgosaocharide.
 4. Thecomposition of claim 1 wherein the attached saccharide is a neutralsaccharide.
 5. The composition of claim 1, wherein the saccharide isselected from the group consisting of lactose and maltose.
 6. Thecomposition of claim 1 wherein the attached sacdiaride is adisaccharide.
 7. The composition of claim 1 wherein a proportion of thelipids having an attached saccharide are covalently crosslinked to otherlipids in the sheet.
 8. The composition of claim 1 wherein a proportionof lipids having an attached saccharide are not covalenty crosslinked toother lipids in the sheet.
 9. The composition of claim 1, wherein aproportion of the lipids in the lipid sheet have a first attachedsaccharide, and a separate proportion of the lipids in the lipid sheethave a second attached saccharide that is different from the first. 10.The composition of claim 1, wherein the first attached saccharide isfucose and the second attached saccharide is a sulfated or acidicmonosaccharide.
 11. The composition of claim 1, wherein the acid groupis a carboxylic acid.
 12. The composition of claim 1, wherein the acidgroup is a negatively charged sulfate or phosphate group.
 13. Thecomposition of claim 1, wherein the lipid sheet is part of the lipidbilayer of a liposome.
 14. The composition of claim 1, wherein thelipids each contain a single aliphatic hydrocarbon.
 15. The compositionof claim 1, wherein the composition inhibits binding of the ligand tothe selectin.
 16. The composition of claim 1, wherein the compositionhas a 50% inhibition concentration (IC₅₀) that is 10² -fold lower thanthat of monomer sLe^(x).
 17. The composition of claim 1, wherein thecomposition has a 50% inhibition concentration (IC₅₀) that is 10⁴ -foldlower than that of monomer sLe^(x).
 18. The composition of claim 1,wherein the composition has an IC₅₀ in a selectin-to-cell binding assayof less than 100 nM.
 19. The composition of claim 1, wherein theselectin is P-selectin.
 20. The composition of claim 1, wherein theselectin is L-selectin.